Appearance of drug resistance-associated mutations in human immunodeficiency virus type 1 protease and reverse transcriptase derived from drug-treated Indonesian patients.

PubMed

Khairunisa, Siti Qamariyah; Kotaki, Tomohiro; Witaningrum, Adiana Mutamsari; Yunifiar M, Muhammad Qushai; Sukartiningrum, Septhia Dwi; Nasronudin; Kameoka, Masanori

2015-02-01

Although HIV-1 drug resistance is a major obstacle in Indonesia, information on drug resistance is limited. In this study, the viral subtype and appearance of drug resistance mutations in the HIV-1 protease (PR) and reverse transcriptase (RT) genes were determined among drug-treated, HIV-1-infected patients in Surabaya. HIV-1 patients who received antiretroviral therapy (ART) more than 2 years were randomly recruited regardless of the viral load or ART failure. Fifty-eight HIV-1 PR genes and 53 RT genes were sequenced. CRF01_AE viruses were identified as the predominant strain. Major drug resistance mutations were not detected in the PR genes. In contrast, 37.7% (20/53) of the participants had one or more major drug resistance mutations in the RT genes, predominantly M184V (28.3%), K103N (11.3%), and thymidine analogue mutations (TAMs) (20.8%). The high prevalence of drug resistance mutations in RT genes indicated the necessity of monitoring the effectiveness of ART in Indonesia.

Schizosaccharomyces pombe Retrotransposon Tf2 Mobilizes Primarily through Homologous cDNA Recombination

PubMed Central

Hoff, Eleanor F.; Levin, Henry L.; Boeke, Jef D.

1998-01-01

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5′ untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons. PMID:9774697

Clinical and virologic follow-up in perinatally HIV-1-infected children and adolescents in Madrid with triple-class antiretroviral drug-resistant viruses.

PubMed

Rojas Sánchez, P; de Mulder, M; Fernandez-Cooke, E; Prieto, L; Rojo, P; Jiménez de Ory, S; José Mellado, M; Navarro, M; Tomas Ramos, J; Holguín, Á

2015-06-01

Drug resistance mutations compromise the success of antiretroviral treatment in human immunodeficiency virus type 1 (HIV-1)-infected children. We report the virologic and clinical follow-up of the Madrid cohort of perinatally HIV-infected children and adolescents after the selection of triple-class drug-resistant mutations (TC-DRM). We identified patients from the cohort carrying HIV-1 variants with TC-DRM to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors according to IAS-USA-2013. We recovered pol sequences or resistance profiles from 2000 to 2011 and clinical-immunologic-virologic data from the moment of TC-DRM detection until December 2013. Viruses harbouring TC-DRM were observed in 48 (9%) of the 534 children and adolescents from 2000 to 2011, rising to 24.4% among those 197 with resistance data. Among them, 95.8% were diagnosed before 2003, 91.7% were Spaniards, 89.6% carried HIV-1-subtype B and 75% received mono/dual therapy as first regimen. The most common TC-DRM present in ≥50% of them were D67NME, T215FVY, M41L and K103N (retrotranscriptase) and L90M (protease). The susceptibility to darunavir, tipranavir, etravirine and rilpivirine was 67.7%, 43.7%, 33.3% and 33.3%, respectively, and all reported high resistance to didanosine, abacavir and nelfinavir. Despite the presence of HIV-1 resistance mutations to the three main antiretroviral families in our paediatric cohort, some drugs maintained their susceptibility, mainly the new protease inhibitors (tipranavir and darunavir) and nonnucleoside reverse transcriptase inhibitors (etravirine and rilpivirine). These data will help to improve the clinical management of HIV-infected children with triple resistance in Spain. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

High Rates of Baseline Drug Resistance and Virologic Failure Among ART-naive HIV-infected Children in Mali.

PubMed

Crowell, Claudia S; Maiga, Almoustapha I; Sylla, Mariam; Taiwo, Babafemi; Kone, Niaboula; Oron, Assaf P; Murphy, Robert L; Marcelin, Anne-Geneviève; Traore, Ban; Fofana, Djeneba B; Peytavin, Gilles; Chadwick, Ellen G

2017-11-01

Limited data exist on drug resistance and antiretroviral treatment (ART) outcomes in HIV-1-infected children in West Africa. We determined the prevalence of baseline resistance and correlates of virologic failure (VF) in a cohort of ART-naive HIV-1-infected children <10 years of age initiating ART in Mali. Reverse transcriptase and protease genes were sequenced at baseline (before ART) and at 6 months. Resistance was defined according to the Stanford HIV Genotypic Resistance database. VF was defined as viral load ≥1000 copies/mL after 6 months of ART. Logistic regression was used to evaluate factors associated with VF or death >1 month after enrollment. Post hoc, antiretroviral concentrations were assayed on baseline samples of participants with baseline resistance. One-hundred twenty children with a median age 2.6 years (interquartile range: 1.6-5.0) were included. Eighty-eight percent reported no prevention of mother-to-child transmission exposure. At baseline, 27 (23%), 4 (3%) and none had non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor or protease inhibitor resistance, respectively. Thirty-nine (33%) developed VF and 4 died >1 month post-ART initiation. In multivariable analyses, poor adherence [odds ratio (OR): 6.1, P = 0.001], baseline NNRTI resistance among children receiving NNRTI-based ART (OR: 22.9, P < 0.001) and protease inhibitor-based ART initiation among children without baseline NNRTI resistance (OR: 5.8, P = 0.018) were significantly associated with VF/death. Ten (38%) with baseline resistance had detectable levels of nevirapine or efavirenz at baseline; 7 were currently breastfeeding, but only 2 reported maternal antiretroviral use. Baseline NNRTI resistance was common in children without reported NNRTI exposure and was associated with increased risk of treatment failure. Detectable NNRTI concentrations were present despite few reports of maternal/infant antiretroviral use.

Recent advances in the development of next generation non-nucleoside reverse transcriptase inhibitors.

PubMed

Tarby, Christine M

2004-01-01

Since their discovery, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become one of the cornerstones of highly active anti-retroviral therapy (HAART). Currently, three NNRTI agents, efavirenz, nevirapine and delavirdine are commercially available. Efavirenz and nevirapine, used in combination with nucleoside reverse transcriptase inhibitors (NRTIs), provide durable regimens with efficacy comparable to protease inhibitor (PI) containing therapies. When virological failure occurs following treatment with an NNRTI, the resistance mutations can confer reduced sensitivity to the entire agent class. Therefore, the strategy for the development of next generation NNRTIs has been to focus on compounds which have improved potencies against the clinically relevant viral mutants. Agents with improved virological profiles and which maintain the ease of administration and favorable safety profiles of the current agents should find use in anti-retroviral naïve patients as well as in components of salvage regimens in the anti-retroviral experienced patient. This review summarizes the recent developments with compounds in clinical trials as of January 2002 as well as to summarize information on new agents appearing in the primary and patent literature between January 2001 and December 2002.

In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

PubMed Central

La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

2001-01-01

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

A decade of HIV-1 drug resistance in the United States: trends and characteristics in a large protease/reverse transcriptase and co-receptor tropism database from 2003 to 2012.

PubMed

Paquet, Agnes C; Solberg, Owen D; Napolitano, Laura A; Volpe, Joseph M; Walworth, Charles; Whitcomb, Jeannette M; Petropoulos, Christos J; Haddad, Mojgan

2014-01-01

Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database. Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated. Within 62,397 resistant viruses reported from 2003 to 2012, we observed a decreasing trend in the prevalence of three-class resistance (from 25% to 9%) driven by decreased resistance to PIs (43% to 21%) and NRTIs (79% to 57%), while observing a slight increase in NNRTI resistance (68% to 75%). The prevalence of CXCR4-mediated entry among tropism testing samples (n=52,945) declined over time from 47% in 2007 to 40% in 2012. A higher proportion of CXCR4-tropic viruses was observed within samples with three-class resistance (50%) compared with the group with no resistance (36%). Decreased prevalence of three-class resistance and increased prevalence of one-class resistance was observed within samples reported between 2003 and 2012. The fraction of CXCR4-tropic viruses has decreased over time; however, CXCR4 usage was more prevalent among multi-class-resistant samples, which may be due to the more advanced disease stage of treatment-experienced patients. These trends have important implications for clinical practice and future drug discovery and development.

Do non-nucleoside reverse transcriptase inhibitors contribute to lipodystrophy?

PubMed

Nolan, David

2005-01-01

Lipodystrophy complications, including lipoatrophy (pathological fat loss) and metabolic complications, have emerged as important long-term toxicities associated with antiretroviral therapy in the current era. The wealth of data that has accumulated over the past 6 years has now clarified the contribution of specific antiretroviral drugs to the risk of these clinical endpoints, with evidence that lipoatrophy is strongly associated with the choice of nucleoside reverse transcriptase inhibitor therapy (specifically, stavudine and to a lesser extent zidovudine). The aetiological basis of metabolic complications of antiretroviral therapy has proven to be complex, in that the risk appears to be modulated by a number of lifestyle factors that have made the metabolic syndrome highly prevalent in the general population, with additional contributions from HIV disease status itself, as well as from individual drugs within the HIV protease inhibitor class. The currently licensed non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs, efavirenz and nevirapine, have been proven to have a favourable safety profile in terms of lipodystrophy complications. However, it must be noted that NNRTI drugs also have individual toxicity profiles that must be accounted for when considering and/or monitoring their use in the treatment of HIV infection.

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

PubMed

Patick, A K; Boritzki, T J; Bloom, L A

1997-10-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

PubMed Central

Patick, A K; Boritzki, T J; Bloom, L A

1997-01-01

Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity. PMID:9333041

Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea.

PubMed

Cercós, M; Santamaría, S; Carbonell, J

1999-04-01

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Fragment Screening and HIV Therapeutics

PubMed Central

Bauman, Joseph D.; Patel, Disha; Arnold, Eddy

2013-01-01

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide-range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target. PMID:21972022

Drug resistance mutations in HIV type 1 isolates from naive patients eligible for first line antiretroviral therapy in JJ Hospital, Mumbai, India.

PubMed

Deshpande, Alake; Karki, Surendra; Recordon-Pinson, Patricia; Fleury, Herve J

2011-12-01

More than 50 HIV-1-infected patients, naive of antiretroviral therapy (ART) but eligible for first line ART in JJ Hospital, Mumbai, India were investigated for surveillance drug resistance mutations (SDRMs); all but one virus belonged to subtype C; we could observe SDRMs to nonnucleoside reverse transcriptase inhibitors and protease inhibitors in 9.6% of the patients.

Approved Antiviral Drugs over the Past 50 Years

PubMed Central

2016-01-01

SUMMARY Since the first antiviral drug, idoxuridine, was approved in 1963, 90 antiviral drugs categorized into 13 functional groups have been formally approved for the treatment of the following 9 human infectious diseases: (i) HIV infections (protease inhibitors, integrase inhibitors, entry inhibitors, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and acyclic nucleoside phosphonate analogues), (ii) hepatitis B virus (HBV) infections (lamivudine, interferons, nucleoside analogues, and acyclic nucleoside phosphonate analogues), (iii) hepatitis C virus (HCV) infections (ribavirin, interferons, NS3/4A protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors), (iv) herpesvirus infections (5-substituted 2′-deoxyuridine analogues, entry inhibitors, nucleoside analogues, pyrophosphate analogues, and acyclic guanosine analogues), (v) influenza virus infections (ribavirin, matrix 2 protein inhibitors, RNA polymerase inhibitors, and neuraminidase inhibitors), (vi) human cytomegalovirus infections (acyclic guanosine analogues, acyclic nucleoside phosphonate analogues, pyrophosphate analogues, and oligonucleotides), (vii) varicella-zoster virus infections (acyclic guanosine analogues, nucleoside analogues, 5-substituted 2′-deoxyuridine analogues, and antibodies), (viii) respiratory syncytial virus infections (ribavirin and antibodies), and (ix) external anogenital warts caused by human papillomavirus infections (imiquimod, sinecatechins, and podofilox). Here, we present for the first time a comprehensive overview of antiviral drugs approved over the past 50 years, shedding light on the development of effective antiviral treatments against current and emerging infectious diseases worldwide. PMID:27281742

Differences in resistance mutations among HIV-1 non-subtype B infections: a systematic review of evidence (1996–2008)

PubMed Central

2009-01-01

Ninety percent of HIV-1-infected people worldwide harbour non-subtype B variants of HIV-1. Yet knowledge of resistance mutations in non-B HIV-1 and their clinical relevance is limited. Although a few reviews, editorials and perspectives have been published alluding to this lack of data among non-B subtypes, no systematic review has been performed to date. With this in mind, we conducted a systematic review (1996–2008) of all published studies performed on the basis of non-subtype B HIV-1 infections treated with antiretroviral drugs that reported genotype resistance tests. Using an established search string, 50 studies were deemed relevant for this review. These studies reported genotyping data from non-B HIV-1 infections that had been treated with either reverse transcriptase inhibitors or protease inhibitors. While most major resistance mutations in subtype B were also found in non-B subtypes, a few novel mutations in non-B subtypes were recognized. The main differences are reflected in the discoveries that: (i) the non-nucleoside reverse transcriptase inhibitor resistance mutation, V106M, has been seen in subtype C and CRF01_AE, but not in subtype B, (ii) the protease inhibitor mutations L89I/V have been reported in C, F and G subtypes, but not in B, (iii) a nelfinavir selected non-D30N containing pathway predominated in CRF01_AE and CRF02_AG, while the emergence of D30N is favoured in subtypes B and D, (iv) studies on thymidine analog-treated subtype C infections from South Africa, Botswana and Malawi have reported a higher frequency of the K65R resistance mutation than that typically seen with subtype B. Additionally, some substitutions that seem to impact non-B viruses differentially are: reverse transcriptase mutations G196E, A98G/S, and V75M; and protease mutations M89I/V and I93L. Polymorphisms that were common in non-B subtypes and that may contribute to resistance tended to persist or become more frequent after drug exposure. Some, but not all, are recognized as minor resistance mutations in B subtypes. These observed differences in resistance pathways may impact cross-resistance and the selection of second-line regimens with protease inhibitors. Attention to newer drug combinations, as well as baseline genotyping of non-B isolates, in well-designed longitudinal studies with long duration of follow up are needed. PMID:19566959

Selection and Characterization of Drug-Resistant Variants of Human Immunodeficiency Virus (AIDS).

DTIC Science & Technology

1995-10-01

on Antiviral Reserach, Santa Fe, New Mexico , 1995. Page 18 APPENDIX Page 19 p - FACTFILE Mutations in HIV-1 Reverse Transcriptase and Protease...including herpes simplex viruses, varicella -zoster Resistance of clinical HIV-1 isolates to foscarnet has not virus, cytomegalovirus (CMV), hepatitis B...This effect of the Tyr-208 substitution was not ob- reported previously for herpes simplex viruses, varicella -zoster served in MT-2 cells, however. virus

Antiretroviral Drug Use in a Cross-Sectional Population Survey in Africa: NIMH Project Accept (HPTN 043).

PubMed

Fogel, Jessica M; Clarke, William; Kulich, Michal; Piwowar-Manning, Estelle; Breaud, Autumn; Olson, Matthew T; Marzinke, Mark A; Laeyendecker, Oliver; Fiamma, Agnès; Donnell, Deborah; Mbwambo, Jessie K K; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J; Eshleman, Susan H

2017-02-01

Antiretroviral (ARV) drug treatment benefits the treated individual and can prevent HIV transmission. We assessed ARV drug use in a community-randomized trial that evaluated the impact of behavioral interventions on HIV incidence. Samples were collected in a cross-sectional survey after a 3-year intervention period. ARV drug testing was performed using samples from HIV-infected adults at 4 study sites (Zimbabwe; Tanzania; KwaZulu-Natal and Soweto, South Africa; survey period 2009-2011) using an assay that detects 20 ARV drugs (6 nucleoside/nucleotide reverse transcriptase inhibitors, 3 nonnucleoside reverse transcriptase inhibitors, and 9 protease inhibitors; maraviroc; raltegravir). ARV drugs were detected in 2011 (27.4%) of 7347 samples; 88.1% had 1 nonnucleoside reverse transcriptase inhibitors ± 1-2 nucleoside/nucleotide reverse transcriptase inhibitors. ARV drug detection was associated with sex (women>men), pregnancy, older age (>24 years), and study site (P < 0.0001 for all 4 variables). ARV drugs were also more frequently detected in adults who were widowed (P = 0.006) or unemployed (P = 0.02). ARV drug use was more frequent in intervention versus control communities early in the survey (P = 0.01), with a significant increase in control (P = 0.004) but not in intervention communities during the survey period. In KwaZulu-Natal, a 1% increase in ARV drug use was associated with a 0.14% absolute decrease in HIV incidence (P = 0.018). This study used an objective, biomedical approach to assess ARV drug use on a population level. This analysis identified factors associated with ARV drug use and provided information on ARV drug use over time. ARV drug use was associated with lower HIV incidence at 1 study site.

Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

PubMed Central

Lai, Ming-Tain; Munshi, Vandna; Touch, Sinoeun; Tynebor, Robert M.; Tucker, Thomas J.; McKenna, Philip M.; Williams, Theresa M.; DiStefano, Daniel J.; Hazuda, Daria J.; Miller, Michael D.

2009-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection. PMID:19289522

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil.

PubMed

Moura, Maria Edileuza Soares; da Guarda Reis, Mônica Nogueira; Lima, Yanna Andressa Ramos; Eulálio, Kelsen Dantas; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2015-05-01

HIV-1 transmitted-drug-resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross-sectional study describes transmitted-dru- resistance and HIV-1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV-1 PR/RT regions sequenced: 44 females and 45 males, 22 self-declared as men who have sex with men. Transmitted-drug-resistance was investigated by CPR tool (Stanford HIV-1 Drug Resistance/SDRM). HIV-1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted-drug-resistance rate was 11.2% (10/89; CI 95%: 5.8-19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8-43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7-29.3%) and 6.8% in women (3/44; CI 95%: 1.8-17.4%). Singleton mutations to protease-inhibitor/PI, nucleoside-reverse-transcriptase-inhibitor/NRTI or non-nucleoside-reverse-transcriptase-inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted-drug-resistance in all populations with risk behaviors may help control AIDS epidemic locally. © 2015 Wiley Periodicals, Inc.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

PubMed Central

Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

2011-01-01

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

[Study of human immunodeficiency virus transmission chains in Andalusia: analysis from baseline antiretroviral resistance sequences].

PubMed

Pérez-Parra, Santiago; Chueca-Porcuna, Natalia; Álvarez-Estevez, Marta; Pasquau, Juan; Omar, Mohamed; Collado, Antonio; Vinuesa, David; Lozano, Ana Belen; García-García, Federico

2015-11-01

Protease and reverse transcriptase HIV-1 sequences provide useful information for patient clinical management, as well as information on resistance to antiretrovirals. The aim of this study is to evaluate transmission events, transmitted drug resistance, and to georeference subtypes among newly diagnosed patients referred to our center. A study was conducted on 693 patients diagnosed between 2005 and 2012 in Southern Spain. Protease and reverse transcriptase sequences were obtained for resistance to cART analysis with Trugene(®) HIV Genotyping Kit (Siemens, NAD). MEGA 5.2, Neighbor-Joining, ArcGIS and REGA were used for subsequent analysis. The results showed 298 patients clustered into 77 different transmission events. Most of the clusters were formed by pairs (n=49), of men having sex with men (n=26), Spanish (n=37), and below 45 years of age (73.5%). Urban areas from Granada, and the coastal areas of Almeria and Granada showed the greatest subtype heterogeneity. Five clusters were formed by more than 10 patients, and 15 clusters had transmitted drug resistance. The study data demonstrate how the phylogenetic characterization of transmission clusters is a powerful tool to monitor the spread of HIV, and may contribute to design correct preventive measures to minimize it. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

PubMed

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-04-01

The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Osteoporosis and osteopenia are not associated with T-cell activation in older cART-treated HIV-infected patients.

PubMed

Krikke, M; Klomberg, R C W; van der Veer, E; Tesselaar, K; Verhaar, H J J; Hoepelman, A I M; Arends, J E

2017-05-01

A higher risk of developing osteopenia/ osteoporosis has been seen in HIV-infected patients. We compared HIV-infected patients, all treated with combination antiretroviral therapy (cART), with a low bone mineral density (BMD) (T-score < -1) to those with a normal BMD (T-score > -1), examining the relation with T-cell activation and bone turnover markers (c-terminal telopeptide (CTX) and procollagen type 1 amino-terminal propeptide (P1NP)). In this single visit pilot study, bone turnover markers, T-cell activation (CD38 + HLA - DR +) and senescence (CD57+) of T cells were measured in patients who had previously undergone dual energy X-ray absorptiometry scanning. All study participants (n = 16) were male, on cART, with a median age of 61 years (IQR 56-66). Nine patients had osteopenia/osteoporosis. When comparing the patients with osteopenia/osteoporosis with those with a normal BMD, no differences in activation and senescence were found. A relation was seen between higher bone formation (P1NP) and patients who were on cART for longer. The median length of cART use was 5.5 years (IQR 4.5-7.8), with all patients on nucleoside reverse transcriptase inhibitors, 88% on tenofovir, 63% on non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 38% on protease inhibitors. Osteopenia/osteoporosis was seen in 100% of the patients on protease inhibitors versus 30% of those on NNRTIs. This study did not find an association between activated T cells and BMD, thus did not explain the higher prevalence of osteopenia/osteoporosis in HIV-infected patients. Interestingly, this small pilot showed that cART might influence BMD, with a possible negative effect for protease inhibitors and a possible protective effect for NNRTIs. These results warrant further investigation.

Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spannaus, Ralf; Bodem, Jochen, E-mail: Jochen.Bodem@vim.uni-wuerzburg.de

2014-04-15

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies.more » The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.« less

Impact of lopinavir-ritonavir exposure in HIV-1 infected children and adolescents in Madrid, Spain during 2000-2014.

PubMed

Rojas Sánchez, Patricia; Prieto, Luis; Jiménez De Ory, Santiago; Fernández Cooke, Elisa; Navarro, Maria Luisa; Ramos, José Tomas; Holguín, África

2017-01-01

The most-used protease-inhibitor in children is Lopinavir-ritonavir (LPV/r), which provides durable suppression of viral load and increases CD4+T-counts. This study describes the virological outcome of the HIV-1-infected paediatric population exposed to LPV/r during 15 years in Spain. Patients from the Madrid Cohort of HIV-1-infected-children and adolescents exposed to LPV/r as different line therapy during 2000-2014 were selected. The baseline epidemiological-clinical features, viral suppression, changes in CD4+T-CD8+T cell counts and drug susceptibility were recorded before and during LPV/r exposure. Drug resistance mutations (DRM) were identified in viruses from samples collected until 2011. We predicted drug susceptibility to 19 antiretrovirals among those carrying DRM using the Stanford's HIVdb Algorithm. A total of 199 (37.3%) of the 534 patients from the cohort were exposed to LPV/r during 2000-2014 in first (group 1), second (group 2) or more line-therapies (group 3). Patients were mainly Spaniards (81.9%), perinatally infected (96.5%) with subtype-B (65.3%) and HIV-diagnosed before year 2000 (67.8%). The mean age at first LPV/r exposure was 9.7 years. After protease-inhibitor exposure, viral suppression was higher in groups 1 and 2 than in group 3. Viral suppression occurred in 87.5%, 68.6% and 64.8% patients from groups 1, 2 and 3, respectively. Among the 64 patients with available resistance data during LPV/r treatment, 27(42.3%) carried DRM to protease-inhibitor, 28 (58.3%) to reverse-transcriptase-inhibitors and 21 (43.7%) to non-reverse-transcriptase-inhibitors. Darunavir/ritonavir, atazanavir-ritonavir and tipranavir/ritonavir presented the highest susceptibility and nelfinavir the lowest. A better lymphocyte recovering occurred when protease-inhibitor was taken as part of a first-line regimen and a higher number of patients reached viral suppression. The least compromised antiretrovirals for rescue antiretroviral regimens, according to DRM in the LPV/r-exposed-paediatric cohort, were mainly the new protease inhibitors.

Optimal therapies of a virus replication model with pharmacological delays based on reverse transcriptase inhibitors and protease inhibitors

NASA Astrophysics Data System (ADS)

Pei, Yongzhen; Li, Changguo; Liang, Xiyin

2017-11-01

A short delay in the pharmacological effect on account of the time required for drug absorption, distribution, and penetration into target cells after application of any anti-viral drug, is defined by the pharmacological delay (Herz et al 1996 Proc. Natl Acad. Sci. USA 93 7247-51). In this paper, a virus replication model with Beddington-DeAngelis incidence rate and the pharmacological and intracellular delays is presented to describe the treatment to cure the virus infection. The optimal controls represent the efficiency of reverse transcriptase inhibitors and protease inhibitors in suppressing viral production and prohibiting new infections. Due to the fact that both the control and state variables contain delays, we derive a necessary conditions for our optimal problem. Based on these results, numerical simulations are implemented not only to show the optimal therapeutic schedules for different infection and release rates, but also to compare the effective of three treatment programs. Furthermore, comparison of therapeutic effects under different maximum tolerable dosages is shown. Our research indicates that (1) the proper and specific treatment program should be determined according to the infection rates of different virus particles; (2) the optimal combined drug treatment is the most efficient; (3) the appropriate proportion of medicament must be formulated during the therapy due to the non-monotonic relationship between maximum tolerable dosages and therapeutic effects; (4) the therapeutic effect is advantageous when the pharmacological delay is considered.

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

PubMed Central

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P. Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-01-01

Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

HIV Protease Inhibitor Use During Pregnancy Is Associated With Decreased Progesterone Levels, Suggesting a Potential Mechanism Contributing to Fetal Growth Restriction

PubMed Central

Papp, Eszter; Mohammadi, Hakimeh; Loutfy, Mona R.; Yudin, Mark H.; Murphy, Kellie E.; Walmsley, Sharon L.; Shah, Rajiv; MacGillivray, Jay; Silverman, Michael; Serghides, Lena

2015-01-01

Background. Protease inhibitor (PI)–based combination antiretroviral therapy (cART) is administered during pregnancy to prevent perinatal human immunodeficiency virus (HIV) transmission. However, PI use has been associated with adverse birth outcomes, including preterm delivery and small-for-gestational-age (SGA) births. The mechanisms underlying these outcomes are unknown. We hypothesized that PIs contribute to these adverse events by altering progesterone levels. Methods. PI effects on trophoblast progesterone production were assessed in vitro. A mouse pregnancy model was used to assess the impact of PI-based cART on pregnancy outcomes and progesterone levels in vivo. Progesterone levels were assessed in plasma specimens from 27 HIV-infected and 17 HIV-uninfected pregnant women. Results. PIs (ritonavir, lopinavir, and atazanavir) but not nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors reduced trophoblast progesterone production in vitro. In pregnant mice, PI-based cART but not dual-NRTI therapy was associated with significantly lower progesterone levels that directly correlated with fetal weight. Progesterone supplementation resulted in a significant improvement in fetal weight. We observed lower progesterone levels and smaller infants in HIV-infected women receiving PI-based cART, compared with the control group. In HIV-infected women, progesterone levels correlated significantly with birth weight percentile. Conclusions. Our data suggest that PI use in pregnancy may lead to lower progesterone levels that could contribute to adverse birth outcomes. PMID:25030058

HIV protease inhibitor use during pregnancy is associated with decreased progesterone levels, suggesting a potential mechanism contributing to fetal growth restriction.

PubMed

Papp, Eszter; Mohammadi, Hakimeh; Loutfy, Mona R; Yudin, Mark H; Murphy, Kellie E; Walmsley, Sharon L; Shah, Rajiv; MacGillivray, Jay; Silverman, Michael; Serghides, Lena

2015-01-01

Protease inhibitor (PI)-based combination antiretroviral therapy (cART) is administered during pregnancy to prevent perinatal human immunodeficiency virus (HIV) transmission. However, PI use has been associated with adverse birth outcomes, including preterm delivery and small-for-gestational-age (SGA) births. The mechanisms underlying these outcomes are unknown. We hypothesized that PIs contribute to these adverse events by altering progesterone levels. PI effects on trophoblast progesterone production were assessed in vitro. A mouse pregnancy model was used to assess the impact of PI-based cART on pregnancy outcomes and progesterone levels in vivo. Progesterone levels were assessed in plasma specimens from 27 HIV-infected and 17 HIV-uninfected pregnant women. PIs (ritonavir, lopinavir, and atazanavir) but not nucleoside reverse transcriptase inhibitors (NRTIs) or nonnucleoside reverse transcriptase inhibitors reduced trophoblast progesterone production in vitro. In pregnant mice, PI-based cART but not dual-NRTI therapy was associated with significantly lower progesterone levels that directly correlated with fetal weight. Progesterone supplementation resulted in a significant improvement in fetal weight. We observed lower progesterone levels and smaller infants in HIV-infected women receiving PI-based cART, compared with the control group. In HIV-infected women, progesterone levels correlated significantly with birth weight percentile. Our data suggest that PI use in pregnancy may lead to lower progesterone levels that could contribute to adverse birth outcomes. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

WHO Collaborating Centre for Acquired Immunodeficiency Syndrome for the Eastern Mediterranean Regional Office, Faculty of Medicine, Kuwait University, Kuwait.

PubMed

Altawalah, Haya; Al-Nakib, Widad

2014-01-01

In the early 1980s, the World Health Organization (WHO) designated the Virology Unit of the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, a collaborating centre for AIDS for the Eastern Mediterranean Regional Office (EMRO), recognizing it to be in compliance with WHO guidelines. In this centre, research integral to the efforts of WHO to combat AIDS is conducted. In addition to annual workshops and symposia, the centre is constantly updating and renewing its facilities and capabilities in keeping with current and latest advances in virology. As an example of the activities of the centre, the HIV-1 RNA viral load in plasma samples of HIV-1 patients is determined by real-time PCR using the AmpliPrep TaqMan HIV-1 test v2.0. HIV-1 drug resistance is determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene, using the TRUGENE HIV-1 Genotyping Assay on the OpenGene® DNA Sequencing System. HIV-1 subtypes are determined by sequencing the reverse transcriptase and protease regions on the HIV-1 pol gene using the genotyping assays described above. A fundamental program of Kuwait's WHO AIDS collaboration centre is the national project on the surveillance of drug resistance in human deficiency virus in Kuwait, which illustrates how the centre and its activities in Kuwait can serve the EMRO region of WHO. © 2014 S. Karger AG, Basel.

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

PubMed Central

2012-01-01

Abstract In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. Patients and methods A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Results Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. Conclusion The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973 PMID:23044036

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients.

PubMed

Elmi Abar, Aden; Jlizi, Asma; Darar, Houssein Youssouf; Kacem, Mohamed Ali Ben Hadj; Slim, Amine

2012-10-08

In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973.

Maraviroc, as a Switch Option, in HIV-1–infected Individuals With Stable, Well-controlled HIV Replication and R5-tropic Virus on Their First Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Plus Ritonavir-boosted Protease Inhibitor Regimen: Week 48 Results of the Randomized, Multicenter MARCH Study

PubMed Central

Pett, Sarah Lilian; Amin, Janaki; Horban, Andrejz; Andrade-Villanueva, Jaime; Losso, Marcelo; Porteiro, Norma; Sierra Madero, Juan; Belloso, Waldo; Tu, Elise; Silk, David; Kelleher, Anthony; Harrigan, Richard; Clark, Andrew; Sugiura, Wataru; Wolff, Marcelo; Gill, John; Gatell, Jose; Fisher, Martin; Clarke, Amanda; Ruxrungtham, Kiat; Prazuck, Thierry; Kaiser, Rolf; Woolley, Ian; Arnaiz, Juan Alberto; Cooper, David; Rockstroh, Jürgen K.; Mallon, Patrick; Emery, Sean; Kelleher, Anthony; Merlin, Kate; Yeung, Julie; Fsadni, Bertha; Marks, Kat; Suzuki, Kazuo; Rismanto, Nick; Salomon, Horacio; Rubio, Andrea E.; Chibo, Doris; Birch, Chris; Harrigan, Richard; Swenson, Luke; Chan, Dennison; Berg, Thomas; Obermeier, Martin; Kaiser, Rolf; Schuelter, Eugen; Sierra Aragon, Saleta; Luebke, Nadine; Coughlan, Suzie; Dean, Jonathan; Sugiura, Wataru; Iwatani, Yasumasa; Reyes Teran, Gustavo; Avila, Santiago; Ruxrungtham, Kiat; Sirivichayakul, Sunee; Naphassanant, May; Ubolyam, Sasiwimol; Kaye, Steve; Land, Sally; Walker, Sarah; Haubrich, Richard; DeJesus, Edwin; Emery, Sean; Pett, Sarah L.; Tu, Elise; Silk, David; Berthon-Jones, Nisha; Amin, Janaki; Espinosa, Natalie; Courtney-Vega, Kymme; Absar, Noorul; Haskelberg, Hila; Robson, Rose; Donaldson, Anna; Losso, Marcelo; Belloso, Waldo; Guelman, Daniel; Gambardella, Luciana; Valdovinos, Mariana; Gatell, Jose; Arnaiz, Juan; Beleta, Helena; Ramos, Nuria; Targa, Marta; Rockstroh, Jurgen; Späth, Brigitta; Boesecke, Christoph; Engelhardt, Angelika; Fisher, Martin; Perry, Nicky; Clarke, Amanda; Gill, John; Beckthold, Brenda; Clark, Andrew; Drummond, Fraser; Lefevre, Eric; Corr, Sharon; Grant, Carol; Lupo, Sergio; Peroni, Luciana; Italiano, Hospital; Sanchez, Marisa; De Paz Sierra, Mariana; Mejia, Ramos; Losso, Marcelo; Viloria, Guillermo; Parlante, Angel; Bissio, Emiliano; Luchetti, Pablo; Warley, Eduardo; Vieni, Ines; Porteiro, Norma; Vilas, Cecilia; Zarate, Abel; Mayer, Gabriela; Elliot, Julian; Hagenauer, Michelle; Kelley, Mark; Rowling, Diane; Gibson, Abby; Latch, Ngaire; Tabrett, Chantal; Warzywoda, Elizabeth; Cooper, David; Pett, Sarah; MacRae, Karen; Sinclair, Brett; Sinn, Kate; Bloch, Mark; Franic, Teo; Vincent, Trina; Stewart, Natasha; Jayewardene, Avindra; Dwyer, Dominic; Kok, Jennifer; Assam, Delene; Taylor, Janette; King, Patricia; Orth, David; Youds, David; Sowden, David; Johnston, Colleen; Murray, Suzanne; Hehir, Jennifer; Wadham, Samantha; Donohue, William; Thompson, Jill; Garsia, Roger; Turnham, Geoffrey; Madden, Tracey; Woolley, Ian; Gillies, Ainsley; Bryant, Mellissa; Gill, John; Beckthold, Brenda; Walmsley, Sharon; Chan, Warmond; LeBlanc, Roger; Lanteigne, Francois; Mouawad, Rima; Rahal, Ines; Guber, Sergio; Ozturk, Sefika; Smith, Graham; Halpenny, Roberta; Reko, Tatjana; Robinette Hills, Jennifer; Wolff, Marcelo; Prazuck, Thierry; Laurent Hocqueloux, Francois; Wolfgang, Johann; Stephan, Christoph; Ebeling, Franziska; Rockstroh, Juergen; Boesecke, Christoph; Spath, Brigitta; Engelhardt, Angelika; Ole Jensen, Bjorn-Erik; Feind, Cecilie; Meyer-Olson, Dirk; Stoll, Matthias; Hoeper, Kirsten; Beider, Renata; Faetkenheur, Gerd; Thomas Baumgarten, Ellen; Baumgarten, Axel; Ingiliz, Patrick; Wienbreyer, Andreas; Behrendt, Daniela; Nienkarken, Tanja; Stein, Jessen; Jessen, Heiko; Zedlack, Carmen; Mallon, Paddy; Simelane, Sibongile; Assmann, Jennifer; Ghavami-Kia, Bijan; Sugiura, Wataru; Imahashi, Mayumi; Tanabe, Kazue; Yokomaku, Yoshiyuki; Imamura, Junji; Andrade-Villanueva, Jaime; Montes de Oca, Melva; Gonzalez, Lucero; Ponce, David; Mendoza, Andrea; Sierra-Madero, Juan; Sanchez Hernandez, Jesus Eduardo; Jaime Ruiz Ballesteros, Eduardo; del Moral Ponce, Sergio; Mosqueda, Luis; Lopez, Monica; Horban, Andrzej; Ignatowska, Anna; Bakowska, Elzbieta; Pulik, Piotr; Sanz-Moreno, Jose; Paredes, Roger; Puig, Jordi; Domingo, Pere; Gutierrez, Mar; Gatell, Jose; González-Cordón, Ana; Callau, Pili; Lopez Aldeguer, Jose; Cuellar Tovar, Sandra; Leal Noval, Manuel; Rivas, Inmaculada; Delgado-Fernandez, Marcial; Ramon Arribas, Jose; Miguel Castro, Juan; Ruxrungtham, Kiat; Avihingsanon, Anchalee; Maek-a-nantawat, Wirach; Intasan, Jintana; Charoenporn, Walairat; Cuprasitrut, Thidarat; Jaisomkom, Pachuen; Pruksakaew, Kanchana; Winston, Alan; Mullaney, Scott; Fisher, Martin; Clarke, Amanda; Barbour, Lisa; Perry, Nicky; Richardson, Celia; Fox, Julie; Murray, Tammy; Leen, Clifford; Morris, Shelia; Satyajit, Das; Sandhu, Rumun; Tucker, James

2016-01-01

Abstract Background. Alternative combination antiretroviral therapies in virologically suppressed human immunodeficiency virus (HIV)–infected patients experiencing side effects and/or at ongoing risk of important comorbidities from current therapy are needed. Maraviroc (MVC), a chemokine receptor 5 antagonist, is a potential alternative component of therapy in those with R5-tropic virus. Methods. The Maraviroc Switch Study is a randomized, multicenter, 96-week, open-label switch study in HIV type 1–infected adults with R5-tropic virus, virologically suppressed on a ritonavir-boosted protease inhibitor (PI/r) plus double nucleoside/nucleotide reverse transcriptase inhibitor (2 N(t)RTI) backbone. Participants were randomized 1:2:2 to current combination antiretroviral therapy (control), or replacing the protease inhibitor (MVC + 2 N(t)RTI arm) or the nucleoside reverse transcriptase inhibitor backbone (MVC + PI/r arm) with twice-daily MVC. The primary endpoint was the difference (switch minus control) in proportion with plasma viral load (VL) <200 copies/mL at 48 weeks. The switch arms were judged noninferior if the lower limit of the 95% confidence interval (CI) for the difference in the primary endpoint was < −12% in the intention-to-treat (ITT) population. Results. The ITT population comprised 395 participants (control, n = 82; MVC + 2 N(t)RTI, n = 156; MVC + PI/r, n = 157). Baseline characteristics were well matched. At week 48, noninferior rates of virological suppression were observed in those switching away from a PI/r (93.6% [95% CI, −9.0% to 2.2%] and 91.7% [95% CI, −9.6% to 3.8%] with VL <200 and <50 copies/mL, respectively) compared to the control arm (97.6% and 95.1% with VL <200 and <50 copies/mL, respectively). In contrast, MVC + PI/r did not meet noninferiority bounds and was significantly inferior (84.1% [95% CI, −19.8% to −5.8%] and 77.7% [95% CI, −24.9% to −8.4%] with VL <200 and <50 copies/mL, respectively) to the control arm in the ITT analysis. Conclusions. These data support MVC as a switch option for ritonavir-boosted PIs when partnered with a 2-N(t)RTI backbone, but not as part of N(t)RTI-sparing regimens comprising MVC with PI/r. Clinical Trials Registration. NCT01384682. PMID:27048747

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

Five Antiretroviral Drug Class-Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection.

PubMed

Volpe, Joseph M; Ward, Douglas J; Napolitano, Laura; Phung, Pham; Toma, Jonathan; Solberg, Owen; Petropoulos, Christos J; Walworth, Charles M

2015-01-01

Transmitted HIV-1 exhibiting reduced susceptibility to protease and reverse transcriptase inhibitors is well documented but limited for integrase inhibitors and enfuvirtide. We describe here a case of transmitted 5 drug class-resistance in an antiretroviral (ARV)-naïve patient who was successfully treated based on the optimized selection of an active ARV drug regimen. The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report. © The Author(s) 2015.

Lopinavir plus nucleoside reverse-transcriptase inhibitors, lopinavir plus raltegravir, or lopinavir monotherapy for second-line treatment of HIV (EARNEST): 144-week follow-up results from a randomised controlled trial.

PubMed

Hakim, James G; Thompson, Jennifer; Kityo, Cissy; Hoppe, Anne; Kambugu, Andrew; van Oosterhout, Joep J; Lugemwa, Abbas; Siika, Abraham; Mwebaze, Raymond; Mweemba, Aggrey; Abongomera, George; Thomason, Margaret J; Easterbrook, Philippa; Mugyenyi, Peter; Walker, A Sarah; Paton, Nicholas I

2018-01-01

Millions of HIV-infected people worldwide receive antiretroviral therapy (ART) in programmes using WHO-recommended standardised regimens. Recent WHO guidelines recommend a boosted protease inhibitor plus raltegravir as an alternative second-line combination. We assessed whether this treatment option offers any advantage over the standard protease inhibitor plus two nucleoside reverse-transcriptase inhibitors (NRTIs) second-line combination after 144 weeks of follow-up in typical programme settings. We analysed the 144-week outcomes at the completion of the EARNEST trial, a randomised controlled trial done in HIV-infected adults or adolescents in 14 sites in five sub-Saharan African countries (Uganda, Zimbabwe, Malawi, Kenya, Zambia). Participants were those who were no longer responding to non-NRTI-based first-line ART, as assessed with WHO criteria, confirmed by viral-load testing. Participants were randomly assigned to receive a ritonavir-boosted protease inhibitor (lopinavir 400 mg with ritonavir 100 mg, twice per day) plus two or three clinician-selected NRTIs (protease inhibitor plus NRTI group), protease inhibitor plus raltegravir (400 mg twice per day; protease inhibitor plus raltegravir group), or protease inhibitor monotherapy (plus raltegravir induction for first 12 weeks, re-intensified to combination therapy after week 96; protease inhibitor monotherapy group). Randomisation was by computer-generated randomisation sequence, with variable block size. The primary outcome was viral load of less than 400 copies per mL at week 144, for which we assessed non-inferiority with a one-sided α of 0·025, and superiority with a two-sided α of 0·025. The EARNEST trial is registered with ISRCTN, number 37737787. Between April 12, 2010, and April 29, 2011, 1837 patients were screened for eligibility, of whom 1277 patients were randomly assigned to an intervention group. In the primary (complete-case) analysis at 144 weeks, 317 (86%) of 367 in the protease inhibitor plus NRTI group had viral loads of less than 400 copies per mL compared with 312 (81%) of 383 in the protease inhibitor plus raltegravir group (p=0·07; lower 95% confidence limit for difference 10·2% vs specified non-inferiority margin 10%). In the protease inhibitor monotherapy group, 292 (78%) of 375 had viral loads of less than 400 copies per mL; p=0·003 versus the protease inhibitor plus NRTI group at 144 weeks. There was no difference between groups in serious adverse events, grade 3 or 4 adverse events (total or ART-related), or events that resulted in treatment modification. Protease inhibitor plus raltegravir offered no advantage over protease inhibitor plus NRTI in virological efficacy or safety. In the primary analysis, protease inhibitor plus raltegravir did not meet non-inferiority criteria. A regimen of protease inhibitor with NRTIs remains the best standardised second-line regimen for use in programmes in resource-limited settings. European and Developing Countries Clinical Trials Partnership (EDCTP), UK Medical Research Council, Instituto de Salud Carlos III, Irish Aid, Swedish International Development Cooperation Agency, Instituto Superiore di Sanita, Merck, ViiV Healthcare, WHO. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Efficacy and durability of nevirapine in antiretroviral drug näive patients.

PubMed

Lange, Joep M A

2003-09-01

Nevirapine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) that was first reported in the scientific literature in 1990. Varying doses of nevirapine (NVP) and a number of regimens containing this NNRTI have been studied in antiretroviral (ARV) näive patients. Four key studies have compared the efficacy and safety of triple drug regimens containing NVP in ARV näive, HIV-1 infected patients. The INCAS study was the first demonstration of how to use NVP in an effective and durable manner: as a component of a triple drug regimen. The COMBINE Study was a comparison of protease inhibitor (PI)-based and NVP-based triple regimens. The Atlantic Study is comparing the safety and efficacy of three triple drug regimens in ARV näive patients. In this study, treatment consists of a divergent drug regimen (PI and nucleoside reverse transcriptase inhibitors, NRTIs) targeting both HIV-1 protease and reverse transcriptase or a convergent regimen targeting reverse transcriptase alone (three NRTIs or two NRTIs plus a NNRTI). A clinical endpoint study (BI 1090) compared the efficacy and durability of multi-drug regimens in ARV näive patients with high baseline plasma HIV-1 RNA levels (pVLs) and low peripheral blood CD4+ lymphocyte counts. Data from these studies confirm that triple regimens containing NVP suppressed viral replication for up to one year, even when the ARV näive patients had low CD4+ cell counts at baseline. Nevirapine-containing regimens suppressed pVLs to < 50 copies/ mL in approximately 50% of patients in the studies discussed (Intent to Treat analyses). Data from 96 weeks of follow up in the Atlantic Study demonstrates that the regimens containing didanosine and stavudine plus indinavir or NVP were significantly more successful in suppressing pVLs to < 50 copies/mL during this period than a regimen composed of these NRTIs and lamivudine (p < or = 0.001). As with other ARV drugs, NVP should always be used as part of a fully suppressive ARV regimen. When used in this way, it is an effective ARV drug, which contributes to durable virological and immunological responses in approximately half of all treated patients. Nevirapine-containing regimens are effective in patients with advanced HIV-1 infection, i.e., low CD4+ cell counts. Data will soon be available from the 2NN Study that compares the efficacy and safety of four different regimens using NVP once daily, NVP twice daily, efavirenz once daily or a combination of NVP and efavirenz. All four arms of the study include a backbone of stavudine and lamivudine.

Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART

PubMed Central

Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C.; Rivera-Morales, Lydia G.; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J.; Ramirez, Teresa J.; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M.; Vazquez-Martinez, Carlos A.; Rodriguez-Padilla, Cristina

2016-01-01

Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM’s in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI. PMID:26807922

Diketo acids derivatives as integrase inhibitors: the war against the acquired immunodeficiency syndrome.

PubMed

Henao-Mejia, Jorge; Góez, Yenny; Patiño, Pablo; Rugeles, Maria T

2006-06-01

Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.

Protecting the fetus against HIV infection: a systematic review of placental transfer of antiretrovirals.

PubMed

McCormack, Shelley A; Best, Brookie M

2014-11-01

Maternal-to-fetal transfer of antiretroviral drugs contributes to prevention of vertical transmission of HIV. This systematic review discusses published studies containing data pertaining to the pharmacokinetics of placental transfer of antiretrovirals in humans, including paired cord and maternal plasma samples collected at the time of delivery as well as ex vivo placental perfusion models. Articles pertaining to placental transfer of antiretrovirals were identified from PubMed, from references of included articles, and from US Department of Health and Human Services Panel on Treatment of HIV-infected Pregnant Women and Prevention of Perinatal Transmission guidelines. Articles from non-human animal models or that had no original maternal-to-fetal transfer data were excluded. PRISMA guidelines were followed. A total of 103 published studies were identified. Data across studies appeared relatively consistent for the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleotide reverse transcriptase inhibitors (NNRTIs), with cord to maternal ratios approaching 1 for many of these agents. The protease inhibitors atazanavir and lopinavir exhibited consistent maternal-to-fetal transfer across studies, although the transfer may be influenced by variations in drug-binding proteins. The protease inhibitors indinavir, nelfinavir, and saquinavir exhibited unreliable placental transport, with cord blood concentrations that were frequently undetectable. Limited data, primarily from case reports, indicate that darunavir and raltegravir provide detectable placental transfer. These findings appear consistent with current guidelines of using two NRTIs plus an NNRTI, atazanavir/ritonavir, or lopinavir/ritonavir to maximize placental transfer as well as to optimally suppress maternal viral load. Darunavir/ritonavir and raltegravir may reasonably serve as second-line agents.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor.

PubMed

Gong, Qingguo; Menon, Lakshmi; Ilina, Tatiana; Miller, Lena G; Ahn, Jinwoo; Parniak, Michael A; Ishima, Rieko

2011-01-01

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors. © 2010 John Wiley & Sons A/S.

FDA approves efavirenz. Food and Drug Administration.

PubMed

Highleyman, L

1998-10-01

The Food and Drug Administration (FDA) approved DuPont Pharma's new non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (Sustiva, DMP-266). Efavirenz has shown promise in trials with over 2000 participants for up to 24 weeks, and early data suggests it may be as effective as protease inhibitors when used in a combination regimen. It is the first anti-HIV drug approved for once-daily dosing. Efavirenz is well tolerated, and the main side effects reported are dizziness, insomnia, abnormal dreams, and skin rash. Efavirenz has been approved for adults and children, but should not be used by pregnant women. Contact information is provided.

Current concepts of metabolic abnormalities in HIV patients: focus on lipodystrophy.

PubMed

Kolter, Donald P

2003-12-01

HIV infection is associated with a number of metabolic abnormalities, including lipodystrophy, a difficult-to-define disorder whose characteristics include hyperlipidemia, insulin resistance, and fat redistribution. Current data suggest that lipodystrophy is caused by multiple factors. Dual-nucleoside reverse transcriptase inhibitor therapy combined with protease inhibitor therapy has been shown to increase the risk of metabolic abnormalities, but susceptibility independent of drug effects has also been shown. While many of the treatments for the broad range of signs and symptoms of lipodystrophy bring about improvements in patient status, none have been demonstrated to bring about a return to baseline levels.

Maraviroc, as a Switch Option, in HIV-1-infected Individuals With Stable, Well-controlled HIV Replication and R5-tropic Virus on Their First Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Plus Ritonavir-boosted Protease Inhibitor Regimen: Week 48 Results of the Randomized, Multicenter MARCH Study.

PubMed

Pett, Sarah Lilian; Amin, Janaki; Horban, Andrejz; Andrade-Villanueva, Jaime; Losso, Marcelo; Porteiro, Norma; Sierra Madero, Juan; Belloso, Waldo; Tu, Elise; Silk, David; Kelleher, Anthony; Harrigan, Richard; Clark, Andrew; Sugiura, Wataru; Wolff, Marcelo; Gill, John; Gatell, Jose; Fisher, Martin; Clarke, Amanda; Ruxrungtham, Kiat; Prazuck, Thierry; Kaiser, Rolf; Woolley, Ian; Arnaiz, Juan Alberto; Cooper, David; Rockstroh, Jürgen K; Mallon, Patrick; Emery, Sean

2016-07-01

Alternative combination antiretroviral therapies in virologically suppressed human immunodeficiency virus (HIV)-infected patients experiencing side effects and/or at ongoing risk of important comorbidities from current therapy are needed. Maraviroc (MVC), a chemokine receptor 5 antagonist, is a potential alternative component of therapy in those with R5-tropic virus. The Maraviroc Switch Study is a randomized, multicenter, 96-week, open-label switch study in HIV type 1-infected adults with R5-tropic virus, virologically suppressed on a ritonavir-boosted protease inhibitor (PI/r) plus double nucleoside/nucleotide reverse transcriptase inhibitor (2 N(t)RTI) backbone. Participants were randomized 1:2:2 to current combination antiretroviral therapy (control), or replacing the protease inhibitor (MVC + 2 N(t)RTI arm) or the nucleoside reverse transcriptase inhibitor backbone (MVC + PI/r arm) with twice-daily MVC. The primary endpoint was the difference (switch minus control) in proportion with plasma viral load (VL) <200 copies/mL at 48 weeks. The switch arms were judged noninferior if the lower limit of the 95% confidence interval (CI) for the difference in the primary endpoint was < -12% in the intention-to-treat (ITT) population. The ITT population comprised 395 participants (control, n = 82; MVC + 2 N(t)RTI, n = 156; MVC + PI/r, n = 157). Baseline characteristics were well matched. At week 48, noninferior rates of virological suppression were observed in those switching away from a PI/r (93.6% [95% CI, -9.0% to 2.2%] and 91.7% [95% CI, -9.6% to 3.8%] with VL <200 and <50 copies/mL, respectively) compared to the control arm (97.6% and 95.1% with VL <200 and <50 copies/mL, respectively). In contrast, MVC + PI/r did not meet noninferiority bounds and was significantly inferior (84.1% [95% CI, -19.8% to -5.8%] and 77.7% [95% CI, -24.9% to -8.4%] with VL <200 and <50 copies/mL, respectively) to the control arm in the ITT analysis. These data support MVC as a switch option for ritonavir-boosted PIs when partnered with a 2-N(t)RTI backbone, but not as part of N(t)RTI-sparing regimens comprising MVC with PI/r. NCT01384682. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Executive summary of the GeSIDA/National AIDS Plan consensus document on antiretroviral therapy in adults infected by the human immunodeficiency virus (updated January 2014).

PubMed

Berenguer, Juan; Polo, Rosa; Lozano, Fernando; López Aldeguer, José; Antela, Antonio; Arribas, José Ramón; Asensi, Víctor; Blanco, José Ramón; Clotet, Bonaventura; Domingo, Pere; Galindo, María José; Gatell, José María; González-García, Juan; Iribarren, José Antonio; Locutura, Jaime; López, Juan Carlos; Mallolas, Josep; Martínez, Esteban; Miralles, Celia; Miró, José M; Moreno, Santiago; Palacios, Rosario; Pérez Elías, María Jesús; Pineda, Juan Antonio; Podzamczer, Daniel; Portilla, Joaquín; Pulido, Federico; Ribera, Esteban; Riera, Melchor; Rubio, Rafael; Santos, Jesús; Sanz, Jesús; Tuset, Montserrat; Vidal, Francesc; Rivero, Antonio

2014-01-01

In this update, antiretroviral therapy (ART) is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The strength and grade of the recommendation varies with clinical circumstances, number of CD4 cells, comorbid conditions and prevention of transmission of HIV. The objective of ART is to achieve an undetectable plasma viral load. Initial ART should always comprise a combination of 3 drugs, including 2 nucleoside reverse transcriptase inhibitors and a third drug from a different family (non-nucleoside reverse transcriptase inhibitor, protease inhibitor, or integrase inhibitor). This update presents the causes and criteria for switching ART in patients with undetectable plasma viral load and in cases of virological failure. An update is also provided for the specific criteria for ART in special situations (acute infection, HIV-2 infection, and pregnancy) and with comorbid conditions (tuberculosis or other opportunistic infections, kidney disease, liver disease, and cancer). Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

[Use of Nadis(®) software to improve adverse drug reaction reporting of antiretroviral drugs: experience in south west of France (midi-pyrénées)].

PubMed

Pochard, Liselotte; Hauviller, Laurent; Cuzin, Lise; Eyvrard, Fréderic; Sommet, Agnès; Montastruc, Jean-Louis; Bagheri, Haleh

2014-01-01

To study the value of the module of pharmacovigilance in Nadis® to improve the antiretroviral (ARV) drugs-induced adverse drug reactions (ADRs) reporting. We collected the ADRs reported for 17 months from November 2010 until April 2012. Following data were recorded: characteristics of patients, ADRs, ARV drugs. The number of ADRs was compared to those collected in the same period (17 months) before use of Nadis®. The 119 ADRs reported (an increase of 183%) for 109 patients ADRs were mainly gastrointestinal (21.8%) followed by renal (20.2%), neuro-psychiatric (16.8%), hepatic (13.5%), cutaneous (8.4%), metabolic (6.7%) and others (12.6%). The repartition of ARV drugs was: nucleoside (31.8%), nucleotide (13.6%) reverse transcriptase inhibitors respectively, non-nucleoside reverse transcriptase inhibitors (13.1%), protease inhibitors (36.4%), and integrase inhibitors (5.1%). Our results show the improvement of ARV-induced ADRs reporting by Nadis® which could be used to reduce the rate of under-reporting in patients exposed to these drugs. © 2014 Société Française de Pharmacologie et de Thérapeutique.

Transmitted drug resistance is still low in newly diagnosed human immunodeficiency virus type 1 CRF06_cpx-infected patients in Estonia in 2010.

PubMed

Avi, Radko; Huik, Kristi; Pauskar, Merit; Ustina, Valentina; Karki, Tõnis; Kallas, Eveli; Jõgeda, Ene-Ly; Krispin, Tõnu; Lutsar, Irja

2014-03-01

The presence of transmitted drug resistance (TDR) in treatment-naive HIV-1-positive subjects is of concern, especially in the countries of the former Soviet Union in which the number of subjects exposed to antiretrovirals (ARV) has exponentially increased during the past decade. We assessed the rate of TDR among newly diagnosed subjects in Estonia in 2010 and compared it to that in 2008. The study included 325 subjects (87% of all subjects tested HIV positive from January 1 to December 31, 2010). Of the 244 sequenced viral genomic RNA in the reverse transcriptase (RT) region 214 were CRF06_cpx, nine were subtype A1, three (one each) were subtype B and subtype C, CRF02_AG, and CRF03_AB; 15 viruses remained unclassified as putative recombinant forms between CRF06_cpx and subtype A1. HIV-1 TDR mutations in 2010 and 2008 (n=145) occurred at similar frequency in 4.5% (95% CI 2.45; 7.98) and 5.5% (95% CI 1.8; 9.24) of the patients, respectively. In 2010, 2.5% (6/244) of the sequences harbored nonnucleoside reverse transcriptase inhibitor (NNRTI) (K103N and K101E), 1.6% (4/244) nucleoside reverse transcriptase inhibitor (NRTI) (M41L, M184I, and K219E), and 0.4% (1/244) protease inhibitor (PI) (V82A) mutations. Our findings indicate that in spite of the increased consumption of ARVs the rate of TDR in Estonia has remained unchanged over the past 3 years. Similar stabilizing or even decreasing trends have been described in Western Europe and North America albeit at higher levels and in different socioeconomic backgrounds.

Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

PubMed Central

Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

2014-01-01

Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

The mechano-chemistry of a monomeric reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei

2017-01-01

Abstract Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex. PMID:29165701

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

PubMed Central

Rice, W G; Schaeffer, C A; Graham, L; Bu, M; McDougal, J S; Orloff, S L; Villinger, F; Young, M; Oroszlan, S; Fesen, M R

1993-01-01

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle. Images Fig. 3 Fig. 4 PMID:7692451

The MONET trial: darunavir/ritonavir with or without nucleoside analogues, for patients with HIV RNA below 50 copies/ml.

PubMed

Arribas, Jose R; Horban, Andrzej; Gerstoft, Jan; Fätkenheuer, Gerdt; Nelson, Mark; Clumeck, Nathan; Pulido, Federico; Hill, Andrew; van Delft, Yvon; Stark, Thomas; Moecklinghoff, Christiane

2010-01-16

In virologically suppressed patients, darunavir-ritonavir (DRV/r) monotherapy could maintain virological suppression similarly to DRV/r and two nucleosides. Two hundred and fifty-six patients with HIV RNA less than 50 copies/ml for over 24 weeks on current antiretrovirals [non-nucleoside reverse transcriptase inhibitor (NNRTI)-based (43%), or protease inhibitor-based (57%)], switched to DRV/r 800/100 mg once daily, either as monotherapy (n = 127) or with two nucleoside reverse transcriptase inhibitors (NRTIs) (n = 129). Treatment failure was defined as two consecutive HIV RNA levels above 50 copies/ml (TLOVR) by week 48, or switches off study treatment. The trial had 80% power to show noninferiority for the monotherapy arm (delta = -12%). Patients were 81% male and 91% Caucasian, with mean age 44 years, and CD4 cell count of 574 cells/microl. In the primary efficacy analysis, HIV RNA less than 50 copies/ml by week 48 (per protocol) was 86.2 versus 87.8% in the monotherapy and triple therapy arms; by intent-to-treat switch equals failure, efficacy was 84.3 versus 85.3%; by a switch-included analysis, efficacy was 93.5 versus 95.1%: all three comparisons showed noninferior efficacy for DRV/r monotherapy. CD4 cell counts remained stable during the trial in both arms. One patient per arm showed at least one protease inhibitor mutation, and one patient in the triple therapy arm showed an NRTI mutation. Nine patients per arm discontinued randomized treatment for either adverse events or other reasons. No new or unexpected safety signals were detected. In this study for patients with HIV RNA less than 50 copies/ml on other antiretrovirals at baseline, switching to DRV/r monotherapy showed noninferior efficacy versus triple antiretroviral therapy.

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study.

PubMed

Prosperi, Mattia C F; Mackie, Nicola; Di Giambenedetto, Simona; Zazzi, Maurizio; Camacho, Ricardo; Fanti, Iuri; Torti, Carlo; Sönnerborg, Anders; Kaiser, Rolf; Codoñer, Francisco M; Van Laethem, Kristel; Bansi, Loveleen; van de Vijver, David A M C; Geretti, Anna Maria; De Luca, Andrea

2011-08-01

Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL. A total of 16 511 HIV-1 reverse transcriptase and protease sequences from 11 492 treatment-experienced patients were identified, and linked to clinical data on viral load, CD4 T cell counts and antiretroviral treatment history. Test results from 3162 treatment-naive patients served as controls. Multivariable analysis was employed to identify predictors of reverse transcriptase and protease DRMs. Overall, 2500/16 511 (15.14%) test results were obtained at a viral load <1000 copies/mL. Individuals with viral load levels of 1000-10000 copies/mL showed the highest probability of drug resistance to any drug class. Independently from other measurable confounders, treatment-experienced patients showed a trend for DRMs and other mutations to decrease at viral load levels <500 copies/mL. Genotypic testing at low viral load may identify emerging antiretroviral drug resistance at an early stage, and thus might be successfully employed in guiding prompt management strategies that may reduce the accumulation of resistance and cross-resistance, viral adaptive changes, and larger viral load increases.

Transmitted Antiretroviral Drug Resistance in Newly HIV-Infected and Untreated Patients in Ségou and Bamako, Mali

PubMed Central

Fofana, Djeneba Bocar; Maiga, Aichatou Chehy; Diallo, Fodie; Ait-Arkoub, Zaina; Daou, Fatoumata; Cisse, Mamadou; Sarro, Yaya dit Sadio; Oumar, Aboubacar Alassane; Sylla, Aliou; Katlama, Christine; Taiwo, Babafemi; Murphy, Robert; Tounkara, Anatole; Marcelin, Anne-Genevieve; Calvez, Vincent

2013-01-01

Abstract The WHO recommends regular surveillance for transmitted antiretroviral drug-resistant viruses in HIV antiretroviral treatment (ART)-naive patients in resource-limited settings. This study aimed to assess the prevalence of mutations associated with resistance in ART-naive patients newly diagnosed with HIV in Bamako and Ségou in Mali. HIV-positive patients who never received ART were recruited in Bamako and Ségou, Mali. The reverse transcriptase (RT) and protease (PR) genes of these patients were sequenced by the “ViroSeq” method. Analysis and interpretation of the resistance were made according to the WHO 2009 list of drug resistance mutations. In all, 51/54 (94.4%) sample patients were sequenced. The median age (IQR) of our patients was 24 (22–27) years and the median CD4 count was 380 (340–456) cells/mm3. The predominant subtype was recombinant HIV-1 CRF02_AG (66.7%) followed by CRF06_cpx (12%) and CRF09_cpx (4%). Four patients had mutations associated with resistance, giving an overall prevalence of resistance estimated at 7.9%. There were two (4%) patients with nucleoside reverse transcriptase inhibitor (NRTI) mutations (one M184V and one T215Y), two (4%) with non-NRTI mutations (two K103N), and one (2%) with a protease inhibitor mutation (one I54V). The prevalence of primary resistance in newly infected patients in Mali is moderate (7.9%). This indicates that the standard NNRTI-based first-line regimen used in Mali is suboptimal for some patients. This study should be done regularly to inform clinical practice. PMID:22823755

HIV Drug Resistance Testing in a Resource Limited Setting with High Viral Diversity: The First Twenty Eight Months Experience.

PubMed

Ngo-Malabo, Elodie Teclaire; Ngoupo, Paul Alain; Sadeuh-Mba, Serge Alain; Akongnwi, Emmanuel; Banaï, Robert; Ngono, Laure; Bilong-Bilong, Charles Felix; Kfutwah, Anfumbom; Njouom, Richard

2017-01-01

First line antiretroviral therapy in a resource-limited setting consists of nucleotide and non-nucleotide reverse transcriptase inhibitors. Protease inhibitors are the hub of second line therapy. The decision to change antiretroviral therapy for a patient is frequently presumptive because of the lack of genotypic resistance tests in routine follow-up. We describe here the resistance profiles observed in patients with varying terms of antiretroviral therapy in Cameroon after implementation of HIV genotypic resistance testing in routine practice. HIV genotypic resistance testing was carried out on consecutive samples received between August 2013 and November 2015. Protease (Prot) and reverse transcriptase (Rt) genes of the HIV genome were amplified, sequenced and analyzed for drug resistance mutations following the algorithm set up by the French National Agency for research on HIV/AIDS and viral hepatitis. Specimens from a total of 167 patients infected with non-B HIV subtypes were received during the study period. Overall 61.7% patients had viral loads of more than 3log copies/ml, suggesting treatment failure. Among the 72 patients on first line, 56 (77.8%) were resistant to Lamivudine, 57 (79.1%) to Efavirenz and 58 (80.6%) to Nevirapine. Overall, more patients (75.0%) on first line antiretroviral therapy harbored multi-drug resistance compared to their counterparts on second line (25.8%). This study revealed that a group of patients with antiretroviral therapy failure harbored multi-drug resistance mutations related to the majority of drugs in the first line regimen. Therefore, HIV resistance testing could be a useful tool to improve HIV care in resource limited settings like Cameroon where treatment options are limited. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

PubMed

Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

2015-04-24

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

Initial therapy with protease inhibitor-sparing regimens: evaluation of nevirapine and delavirdine.

PubMed

Conway, B

2000-06-01

We have compared the results (on-treatment analyses) of 2 randomized clinical trials of protease inhibitor-sparing regimens in drug-naive patients. In the INCAS (Italy, Netherlands, Canada, Australia) study, the mean decrease in plasma viral load over 52 weeks was 2.2 log(10) copies/mL in 40 patients who were receiving zidovudine/didanosine/nevirapine (18 [45%] had maximal suppression), with a mean increase in CD4 T cell counts of 139 cells/microL. In protocol 0021 Part II, the mean decrease in plasma viral load over 52 weeks was 2.1 log(10) copies/mL in 34 patients who were receiving zidovudine/lamivudine/delavirdine (20 [59%] had maximal suppression), with a mean increase in CD4 T cell counts of 88 cells/microL. The virologic and immunologic efficacy of the 2 triple-drug regimens are similar. Until results of long-term studies are available to establish whether a preferred approach to initial therapy exists, nonnucleoside reverse transcriptase inhibitors may be a valuable alternative to protease inhibitors in the initial therapy of antiretroviral-naive, moderately immunosuppressed patients.

Protease inhibitors as potential therapeutic agents for AIDS.

PubMed

Jamjoom, G A

1991-09-01

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

HIV-1 pol mutation frequency by subtype and treatment experience: extension of the HIVseq program to seven non-B subtypes.

PubMed

Rhee, Soo-Yon; Kantor, Rami; Katzenstein, David A; Camacho, Ricardo; Morris, Lynn; Sirivichayakul, Sunee; Jorgensen, Louise; Brigido, Luis F; Schapiro, Jonathan M; Shafer, Robert W

2006-03-21

HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02. The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation. As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B. HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences.

HIV-1 pol mutation frequency by subtype and treatment experience

PubMed Central

Rhee, Soo-Yon; Kantor, Rami; Katzenstein, David A.; Camacho, Ricardo; Morris, Lynn; Sirivichayakul, Sunee; Jorgensen, Louise; Brigido, Luis F.; Schapiro, Jonathan M.; Shafer, Robert W.

2008-01-01

Objective HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02. Methods The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation. Results As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B. Conclusions HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences. PMID:16514293

High prevalence of bevirimat resistance mutations in protease inhibitor-resistant HIV isolates.

PubMed

Verheyen, Jens; Verhofstede, Chris; Knops, Elena; Vandekerckhove, Linos; Fun, Axel; Brunen, Diede; Dauwe, Kenny; Wensing, Annemarie M J; Pfister, Herbert; Kaiser, Rolf; Nijhuis, Monique

2010-03-13

Bevirimat is the first drug of a new class of antivirals that hamper the maturation of HIV. The objective of this study was to evaluate the sequence variability of the gag region targeted by bevirimat in HIV subtype-B isolates. Of 484 HIV subtype-B isolates, the gag region comprising amino acids 357-382 was sequenced. Of the patients included, 270 were treatment naive and 214 were treatment experienced. In the latter group, 48 HIV isolates harboured mutations associated with reverse transcriptase inhibitor resistance only, and 166 HIV isolates carried mutations associated with protease inhibitor resistance. In the treatment-naive patient population, approximately 30% harboured an HIV isolate with at least one mutation associated with a reduced susceptibility to bevirimat (H358Y, L363M, Q369H, V370A/M/del and T371del). In HIV isolates with protease inhibitor resistance, the prevalence of bevirimat resistance mutations increased to 45%. Accumulation of mutations at four positions in the bevirimat target region, S368C, Q369H, V370A and S373P, was significantly observed. Mutations associated with bevirimat resistance were detected more frequently in HIV isolates with three or more protease inhibitor resistance mutations than in those with less than three protease inhibitor mutations. Reduced bevirimat activity can be expected in one-third of treatment-naive HIV subtype-B isolates and significantly more in protease inhibitor-resistant HIV. These data indicate that screening for bevirimat resistance mutations before administration of the drug is essential.

Antiretroviral therapy modifies the genetic effect of known type 2 diabetes-associated risk variants in HIV-infected women.

PubMed

Frasco, Melissa A; Karim, Roksana; Van Den Berg, David; Watanabe, Richard M; Anastos, Kathryn; Cohen, Mardge; Gange, Stephen J; Gustafson, Deborah R; Liu, Chenglong; Tien, Phyllis C; Mack, Wendy J; Pearce, Celeste L

2014-07-31

Type 2 diabetes mellitus incidence is increased in HIV-infected persons. We examined the associations of diabetes mellitus with known diabetes mellitus-risk alleles from the general population in the context of HIV infection, and explored effect modification by combination antiretroviral therapy (cART). The Women's Interagency HIV Study is a prospective cohort of HIV-infected women. Seventeen European-derived diabetes mellitus-risk polymorphisms were genotyped in the eligible participants of the Women's Interagency HIV Study. Analyses were run separately for non-African Americans (Whites, Hispanics, Asians, and other; n = 378, 49 with incident diabetes mellitus) and African Americans (n = 591, 49 with incident diabetes mellitus). Cox proportional-hazards models were fit to estimate hazard ratios for diabetes mellitus overall and within strata of cART. In non-African Americans, heterogeneity across cART regimen was observed for nine of the 14 polymorphisms (phet < 0.05). One polymorphism was statistically significantly inversely associated with diabetes mellitus risk among women taking two nucleotide reverse transcriptase inhibitors (NRTIs) + non-nucleotide reverse transcriptase inhibitor (NNRTI). Five polymorphisms were statistically significantly associated with diabetes mellitus among women treated with at least two NRTIs + at least one protease inhibitor and one polymorphism was associated with diabetes mellitus among those treated with at least three NRTIs ± NNRTI. The hazard ratio per risk allele for IGF2BP2 rs1470579 was 2.67 (95% confidence interval 1.67-4.31) for women taking cART with at least two NRTIs + at least one protease inhibitor and 2.45 (95% confidence interval 1.08-5.53) in women taking at least three NRTIs ± NNRTI (phet = 2.50 × 10⁻³). No such associations were observed in the African Americans. Genetic susceptibility to diabetes mellitus, based on the variants studied, is substantially elevated among HIV-infected women using cART containing three or more NRTI/protease inhibitor components. A personalized medicine approach to cART selection may be indicated for HIV-infected persons carrying these diabetes mellitus-risk variants.

Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

PubMed Central

Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Haertl, Felix; Matevossian, Edouard; Zernecke, Alma; Eckstein, Hans-Henning; Pelisek, Jaroslav

2014-01-01

Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT–PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT–PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA. PMID:24833013

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Nazar, Muhammad Faizan; Abdullah, Muhammad Imran; Badshah, Amir; Mahmood, Asif; Rana, Usman Ali; Khan, Salah Ud-Din

2015-04-01

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following a convenient route of Robinson annulation, and the molecular structure of these CDs were later confirmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV-Vis spectroscopy and mass spectrometry. All the synthesized compounds were screened theoretically and experimentally against reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the compounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site, contributing high free binding energy (ΔG -8.01 Kcal) and IC50 (0.207 μg/ml), respectively. Further results revealed that the compounds bearing more halogen groups, with additional hydrophobic character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is anticipate that the present study would be very useful for the selection of potential reverse transcriptase inhibitors featuring inclusive pharmacological profiles.

[Mutations of resistance of HIV-1 in previously untreated patients at penitentiary centers of the Autonomous Community of Valencia, Spain. REPRICOVA study].

PubMed

García-Guerrero, Julio; Herrero, Agustín; Vera, Enrique; Almenara, José M; Araújo, Rosa; Saurí, Vicente V; Castellano, Juan C; Fernández-Clemente, Luis; Bedia, Miguel; Llorente, María I; González-Morán, Francisco

2002-03-02

Our purpose was to determine the prevalence of mutations of resistance to nucleoside inhibitors of reverse transcriptase (NIRT) and protease inhibitors (PI) in the HIV-1 genotype of naïve infected subjects in the prisons of the Autonomous Community of Valencia, Spain. Multicentric, descriptive, cross-sectional study of prevalence including a systematic stratified and randomised sampling by centres. Demographic, clinical, virological and immunological data were collected. The HIV gene of protease and transcriptase was studied in peripheral blood plasma samples by means of double PCR amplification and subsequent automatic sequence. Reference: wild strain HXB2. Plasma was obtained from 133 individuals (119 men and 14 women). 117 samples were selected and the rest did not have enough copies for transcription. With regard to NIRT, 7 samples (5.2% of total) showed some mutation of resistance: M41L, D67N, L210W and K219Q, all them secondary to and associated with resistance to zidovudine, abacavir as well as group B multinucleoside-resistance. With regard to PI, only one sample showed a primary mutation, M46I, which was associated with resistance to indinavir. Moreover, a further 41 samples were found to express some secondary mutation. In our series, there was a low number of primary mutations of resistance. These results allow us to exclude the systematic use of resistance tests before an initiation antiretroviral therapy.

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

PubMed

Tamalet, Catherine; Tissot-Dupont, Hervé; Motte, Anne; Tourrès, Christian; Dhiver, Catherine; Ravaux, Isabelle; Poizot-Martin, Isabelle; Dieng, Thérèse; Tomei, Christelle; Bregigeon, Sylvie; Zaegel-Faucher, Olivia; Laroche, Hélène; Aherfi, Sarah; Mokhtari, Saadia; Chaudet, Hervé; Ménard, Amelie; Brouqui, Philippe; Stein, Andreas; Colson, Philippe

2018-05-24

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies. © 2018 Wiley Periodicals, Inc.

Nelfinavir and non-nucleoside reverse transcriptase inhibitor-based salvage regimens in heavily HIV pretreated patients

PubMed Central

Baril, Jean-Guy; Lefebvre, Eric A; Lalonde, Richard G; Shafran, Stephen D; Conway, Brian

2003-01-01

OBJECTIVE: To assess the efficacy of nelfinavir mesylate (NFV) in combination with delavirdine mesylate (DLV) or efavirenz (EFV) and other antiretroviral agents following virological failure on other protease inhibitor (PI)-based regimens. DESIGN: Multicentre, retrospective chart review. METHODS: One hundred-one patients who were naive to both NFV and non-nucleoside reverse transcriptase inhibitors (NNRTIs) and who initiated NFV plus DLV or EFV-based salvage regimens were reviewed. Response to treatment was defined as a reduction in HIV ribonucleic acid (RNA) levels to unquantifiable levels (less than 50 copies/mL, less than 400 copies/mL, less than 500 copies/mL) on at least one occasion after the initiation of salvage therapy. Baseline correlates of response, including prior duration of HIV infection, prior number of regimens, viral load and CD4 cell counts were also evaluated. RESULTS: Patients had a mean duration of HIV infection of 10 years, a mean duration of prior therapy of four years, a median of four prior nucleoside reverse transcriptase inhibitors and a median of two prior PIs. At the time of review the mean duration of salvage therapy was 63.4 weeks. Virological suppression was achieved in 59 (58.4%) patients within a mean of eight weeks and maintained for a mean of 44.9 weeks (the mean follow-up was 78 weeks). Of the non-responders, 16 (38%) achieved a less than 1 log10 decrease in HIV RNA levels. Although there was no association between baseline correlates, response rate (75.7%) was significantly higher in patients with HIV RNA levels of 50,000 copies/mL or lower and CD4 counts greater than 200 cells/mm3. CONCLUSION: NFV/NNRTI-based highly active antiretroviral therapy regimens are an effective therapy in many patients who have experienced virological breakthroughs on at least one prior PI-based regimen. PMID:18159457

High Levels of Transmitted HIV Drug Resistance in a Study in Papua New Guinea.

PubMed

Lavu, Evelyn; Kave, Ellan; Mosoro, Euodia; Markby, Jessica; Aleksic, Eman; Gare, Janet; Elsum, Imogen A; Nano, Gideon; Kaima, Petronia; Dala, Nick; Gurung, Anup; Bertagnolio, Silvia; Crowe, Suzanne M; Myatt, Mark; Hearps, Anna C; Jordan, Michael R

2017-01-01

Papua New Guinea is a Pacific Island nation of 7.3 million people with an estimated HIV prevalence of 0.8%. ART initiation and monitoring are guided by clinical staging and CD4 cell counts, when available. Little is known about levels of transmitted HIV drug resistance in recently infected individuals in Papua New Guinea. Surveillance of transmitted HIV drug resistance in a total of 123 individuals recently infected with HIV and aged less than 30 years was implemented in Port Moresby (n = 62) and Mount Hagen (n = 61) during the period May 2013-April 2014. HIV drug resistance testing was performed using dried blood spots. Transmitted HIV drug resistance was defined by the presence of one or more drug resistance mutations as defined by the World Health Organization surveillance drug resistance mutations list. The prevalence of non-nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 16.1% (95% CI 8.8%-27.4%) and 8.2% (95% CI 3.2%-18.2%) in Port Moresby and Mount Hagen, respectively. The prevalence of nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 3.2% (95% CI 0.2%-11.7%) and 3.3% (95% CI 0.2%-11.8%) in Port Moresby and Mount Hagen, respectively. No protease inhibitor transmitted HIV drug resistance was observed. The level of non-nucleoside reverse transcriptase inhibitor drug resistance in antiretroviral drug naïve individuals recently infected with HIV in Port Moresby is amongst the highest reported globally. This alarming level of transmitted HIV drug resistance in a young sexually active population threatens to limit the on-going effective use of NNRTIs as a component of first-line ART in Papua New Guinea. To support the choice of nationally recommended first-line antiretroviral therapy, representative surveillance of HIV drug resistance among antiretroviral therapy initiators in Papua New Guinea should be urgently implemented.

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil.

PubMed

Ferreira, Ana Cristina G; Coelho, Lara E; Grinsztejn, Eduarda; Jesus, Carlos S de; Guimarães, Monick L; Veloso, Valdiléa G; Grinsztejn, Beatriz; Cardoso, Sandra W

The widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. Patients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal-Wallis and Chi-square tests. Forty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1-30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1-24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. The high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Expression and integrity of dermatopontin in chronic cutaneous wounds: a crucial factor in impaired wound healing.

PubMed

Krishnaswamy, Venkat Raghavan; Manikandan, Mayakannan; Munirajan, Arasambattu Kannan; Vijayaraghavan, Doraiswamy; Korrapati, Purna Sai

2014-12-01

Chronic cutaneous wound (CCW) is a major health care burden wherein the healing process is slow or rather static resulting in anatomical and functional restriction of the damaged tissue. Dysregulated expression and degradation of matrix proteins, growth factors and cytokines contribute to the disrupted and uncoordinated healing process of CCW. Therefore, therapeutic approaches for effective management of CCW should be focused towards identifying and manipulating the molecular defects, such as reduced bioavailability of the pro-healing molecules and elevated activity of proteases. This study essentially deals with assessing the expression and integrity of an extracellular matrix protein, Dermatopontin (DPT), in CCW using real-time quantitative reverse transcriptase PCR and immunological techniques. The results indicate that, despite DPT's high mRNA expression, the protein levels are markedly reduced in both CCW tissue and its exudate. To elucidate the cause for this contradiction in mRNA and protein levels, the stability of DPT is analyzed in the presence of wound exudates and various proteases that are naturally elevated in CCW. DPT was observed to be degraded at higher rates when incubated with certain recombinant proteases or chronic wound exudate. In conclusion, the susceptibility of DPT protein to specific proteases present at high levels in the wound milieu resulted in the degradation of DPT, thus leading to impaired healing response in CCW.

Immunovirological response to triple nucleotide reverse-transcriptase inhibitors and ritonavir-boosted protease inhibitors in treatment-naive HIV-2-infected patients: The ACHIEV2E Collaboration Study Group.

PubMed

Benard, Antoine; van Sighem, Ard; Taieb, Audrey; Valadas, Emilia; Ruelle, Jean; Soriano, Vicente; Calmy, Alexandra; Balotta, Claudia; Damond, Florence; Brun-Vezinet, Françoise; Chene, Geneviève; Matheron, Sophie

2011-05-01

Triple nucleoside reverse-transcriptase inhibitors (NRTIs) are recommended by the World Health Organization as first-line regimen in treatment-naïve HIV-2-infected patients. However, ritonavir-boosted protease inhibitor (PI/r)-containing regimens are frequently prescribed. In the absence of previous randomized trials, we retrospectively compared these regimens in observational cohorts. HIV-2-infected patients from 7 European cohorts who started triple NRTI or PI/r since January 1998 were included. Piecewise linear models were used to estimate CD4 cell count and plasma HIV-2 RNA level slopes, differentiating an early phase (until end of month 3) and a second phase (months 4-12). On-treatment analyses censored data at major treatment modification and systematically at month 12. Forty-four patients started triple NRTI therapy and 126 started PI/r therapy. Overall, the median CD4 cell count was 191 cells/mm(3) and the median plasma HIV-2 RNA level was ≥2.7 log(10) copies/ml in 61% of the patients at combination antiretroviral therapy (cART) initiation; the median duration of the first cART was 20 months, not differing between groups. PI/r regimens were associated with better CD4 cell count and HIV-2 RNA level outcomes, compared with NRTI regimens. Estimated CD4 cell count slopes were +6 and +12 cells/mm(3)/month during the early phase (P = .22), and -60 cells/mm(3)/year versus +76 cells/mm(3)/year during the second phase (P = .002), for triple NRTI and PI/r, respectively. Estimated mean HIV-2 RNA levels at month 12 in patients with detectable viremia at cART initiation were 4.0 and 2.2 log(10) copies/ml, respectively (P = .005). In this observational study, PI/r-containing regimens showed superior efficacy over triple NRTI regimens as first-line therapy in HIV-2-infected patients.

Trends in Drug Resistance Prevalence, HIV-1 Variants and Clinical Status in HIV-1-infected Pediatric Population in Madrid: 1993 to 2015 Analysis.

PubMed

Rojas Sánchez, Patricia; Domínguez, Sara; Jiménez De Ory, Santiago; Prieto, Luis; Rojo, Pablo; Mellado, Pepa; Navarro, Marisa; Delgado, Rafael; Ramos, José Tomas; Holguín, África

2018-03-01

The expanded use of long-term antiretroviral treatments in infected children may exacerbate the problem of drug resistance mutations selection, which can compromise treatment efficiency. We describe the temporal trends of HIV drug resistance mutations and the HIV-1 variants during 23 years (1993 to March 2016) in the Madrid cohort of HIV-infected children and adolescents. We selected patients with at least one available HIV-1 pol sequence/genotypic resistance profile, establishing different groups according to the sampling year of first resistance data. We determined the prevalence of transmitted drug resistance mutations or acquired drug resistance mutations (DRM), the drug susceptibility among resistant viruses and HIV-1 variants characterized by phylogeny across time. A total of 245 pediatric patients were selected, being mainly female, Spanish native, perinatally infected and carrying HIV-1 subtype B. At first sampling, most pediatric patients were on antiretroviral therapy and heavily pretreated. During 1993 to 2016, transmitted drug resistance mutations was found in 13 (26%) of 50 naive children [non-nucleoside reverse transcriptase inhibitors (NNRTI), 14.6%; nucleoside reverse transcriptase inhibitors (NRTI), 10.4%; protease inhibitors, 8.7%]. DRM appeared in 139 (73.2%) of 190 pretreated patients (NRTI, 64.5%; NNRTI, 36%; protease inhibitors, 35.1%). DRM to NNRTI was higher in last 5 years. Non-B variants infected 14.5% of children and adolescents of the Madrid Cohort, being mainly intersubtype recombinants (76.5%), including complex unique recombinant strains. They caused 3.4% infections before 2000, rising to 85.7% during 2011 to 2016. Periodic surveillance resistance and molecular epidemiology studies in long-term pretreated HIV-infected pediatric populations are required to optimize treatment regimens. Results will permit a better understanding of long-time dynamics of viral resistance and HIV-1 variants in Spain.

Differences in National Antiretroviral Prescribing Patterns between Black and White Patients with HIV/AIDS, 1996–2006

PubMed Central

Oramasionwu, Christine U.; Brown, Carolyn M.; Lawson, Kenneth A.; Ryan, Laurajo; Skinner, Jeff; Frei, Christopher R.

2011-01-01

Objectives The benefit of improved health outcomes for blacks receiving highly active antiretroviral therapy (HAART) lags behind that of whites. This project therefore sought to determine whether the reason for this discrepancy in health outcomes could be attributed to disparities in use of antiretroviral therapy between black and white patients with HIV. Materials and Methods The 1996–2006 National Hospital Ambulatory Medical Care Surveys were used to identify hospital outpatient visits that documented antiretrovirals. Patients younger than 18 years, of nonblack or nonwhite race, and lacking documentation of antiretrovirals were excluded. A multivariable logistic regression model was constructed with race as the independent variable and use of HAART as the dependent variable. Results Approximately 3 million HIV/AIDS patient visits were evaluated. Blacks were less likely than whites to use HAART and protease inhibitors (odds ratio, 95% CI 0.81 [0.81–0.82] and 0.67 [0.67–0.68], respectively). More blacks than whites used non-nucleoside reverse transcriptase inhibitors (odds ratio, 95% CI 1.18 [1.17–1.18]). In 1996, the crude rates of HAART were relatively low for both black and white cohorts (5% vs 6%). The rise in HAART for blacks appeared to lag behind that of whites for several years, until 2002, when the proportion of blacks receiving HAART slightly exceeded the proportion of whites receiving HAART. In later years, the rates of HAART were similar for blacks and whites (81% vs 82% in 2006). Blacks appeared less likely than whites to use protease inhibitors and more likely than whites to use non-nucleoside reverse transcriptase inhibitors from 2000 to 2004. Conclusions Blacks experienced a lag in the use of antiretrovirals at the beginning of the study; this discrepancy dissipated in more recent years. PMID:22089356

Executive summary of the GESIDA/National AIDS Plan Consensus Document on Antiretroviral Therapy in Adults Infected by the Human Immunodeficiency Virus (Updated January 2016).

PubMed

2016-01-01

In this update, antiretroviral therapy (ART) is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The objective of ART is to achieve an undetectable plasma viral load (PVL). Initial ART should comprise 3 drugs, namely, 2 nucleoside reverse transcriptase inhibitors (NRTI), and 1 drug from another family. Four of the recommended regimens, all of which have an integrase strand transfer inhibitor (INSTI) as the third drug, are considered a preferred regimen; a further 6 regimens, which are based on an INSTI, a non-nucleoside reverse transcriptase inhibitor (NNRTI), or a protease inhibitor boosted with cobicistat or ritonavir (PI/COBI, PI/r), are considered alternatives. The reasons and criteria for switching ART are presented both for patients with an undetectable PVL and for patients who experience virological failure, in which case the rescue regimen should include 3 (or at least 2) drugs that are fully active against HIV. The specific criteria for ART in special situations (acute infection, HIV-2 infection, pregnancy) and comorbid conditions (tuberculosis and other opportunistic infections, kidney disease, liver disease, and cancer) are updated. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Genetic characterization and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China.

PubMed

Guo, Jinlei; Yan, Yong; Zhang, Jiafeng; Ji, Jimei; Ge, Zhijian; Ge, Rui; Zhang, Xiaofei; Wang, Henghui; Chen, Zhongwen; Luo, Jianyong

2017-03-14

The aim of this study was to characterize HIV-1 genotypes and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China. The HIV-1 partial polymerase (pol) genes in 93 of the 99 plasma samples were successfully amplified and analyzed. Phylogenetic analysis revealed the existence of five HIV-1 genotypes, of which the most prevalent genotype was CRF01_AE (38.7%), followed by CRF07_BC (34.4%), CRF08_BC (16.1%), subtype B/B' (5.4%), and CRF55_01B (2.1%). Besides, three types of unique recombination forms (URFs) were also observed, including C/F2/A1, CRF01_AE/B, and CRF08_BC/CRF07_BC. Among 93 amplicons, 46.2% had drug resistance-associated mutations, including 23.7% for protease inhibitors (PIs) mutations, 1.1% for nucleoside reverse transcriptase inhibitors (NRTIs) mutations, and 20.4% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations. Six (6.5%) out of 93 treatment-naive subjects were identified to be resistant to one or more NNRTIs, while resistance to NRTIs or PIs was not observed. Our study showed the genetic diversity of HIV-1 strains circulating in Jiaxing and a relative high proportion of antiretroviral resistance mutations among treatment-naive patients, indicating a serious challenge for HIV prevention and treatment program.

Prevalence of Drug-Resistance Mutations and Non–Subtype B Strains Among HIV-Infected Infants From New York State

PubMed Central

Karchava, Marine; Pulver, Wendy; Smith, Lou; Philpott, Sean; Sullivan, Timothy J.; Wethers, Judith; Parker, Monica M.

2010-01-01

Summary Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001–2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998–1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998–1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non–subtype B strain of HIV. It seems that drug-resistant and non–subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies. PMID:16868498

New Antiretroviral Therapies for Pediatric HIV Infection

PubMed Central

Morris, Jennifer L.; Kraus, Donna M.

2005-01-01

Human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome affect millions of children worldwide. The development of antiretroviral therapy has significantly improved the morbidity and mortality of pediatric patients infected with HIV. Currently, 4 classes of antiretroviral agents exist: nucleoside / nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and entry inhibitors. A total of 21 single-entity antiretroviral agents and 4 co-formulated antiretroviral products hold Food and Drug Administration (FDA) approval for treatment of HIV-1 infection. However, not all of these agents are indicated for use in patients less than 18 years of age. Since the year 2000, 7 new antiretroviral agents (atazanavir, emtricitabine, enfuvirtide, fosamprenavir, lopinavir/ritonavir, tenofovir, and tipranavir) have been approved by the FDA for use in adult patients as part of combination therapy for the treatment of HIV-1 infection. Although only 3 of these newer agents (emtricitabine, enfuvirtide, and lopinavir/ritonavir) are currently FDA approved for use in pediatric patients, pediatric clinical studies of the other 4 new agents are currently underway. The purpose of this article is to review these 7 new antiretroviral agents and describe their roles in the treatment of pediatric HIV infection. For each drug, the following information will be addressed: FDA-approved indication and age groups, clinical efficacy, pharmacokinetics, adverse drug reactions, clinically relevant drug interactions, pediatric and adult dosing, dosage forms, administration, and place in the treatment of pediatric HIV infection. PMID:23118639

Low prevalence of primary HIV resistance in western Massachusetts.

PubMed

Iarikov, Dmitri E; Irizarry-Acosta, Melina; Martorell, Claudia; Hoffman, Robert P; Skiest, Daniel J

2010-01-01

Most studies of primary antiretroviral (ARV) resistance have been conducted in large metropolitan areas with reported rates of 8% to 25%. We collected data on 99 HIV-1-infected antiretroviral-naive patients from several sites in Springfield, MA, who underwent genotypic resistance assay between 2004 and 2008. Only major resistance mutations per International AIDS Society-USA (IAS-USA) drug resistance mutations list were considered. The prevalence of resistance was 5% (5 of 99). Three patients had one nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation: 103N, 103N, and 190A, 1 patient had a protease inhibitor (PI) mutation: 90M; and 1 patient had 3-class resistance with NNRTI: 181C, 190A, PI: 90M, and nucleoside analogue reverse transcriptase inhibitor (NRTI): 41L, 210W. Mean time from HIV diagnosis to resistance testing was shorter in patients with resistance versus those without: 9 (range 0.3-42 months) versus 27 (range 0.1-418 months), P = .11. There was a trend to lower mean CD4 count in those with resistance, 170 versus 318 cells/mm(3), P = .06. No differences were noted in gender, age, HIV risk category, or HIV RNA level. The low prevalence of primary resistance may be explained by differences in demographic and risk factors or may reflect the time from infection to resistance testing. Our findings emphasize the importance of continued resistance surveillance.

An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

PubMed Central

Melnick, Laurence; Yang, Shiow-Shong; Rossi, Rick; Zepp, Charlie; Heefner, Donald

1998-01-01

We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity. PMID:9835523

Antiviral interactions of combinations of highly potent 2,4(1H,3H)-pyrimidinedione congeners and other anti-HIV agents.

PubMed

Hartman, Tracy L; Yang, Lu; Buckheit, Robert W

2011-12-01

Structure-activity relationship evaluation of seventy-four 2,4(1H,3H)-pyrimidinedione derivatives identified seven lead compounds based on anti-HIV-1 potency, extended range of action to include HIV-2, virus entry inhibition, reverse transcriptase inhibition, and lack of cytotoxicity to human cells. The selected pyrimidinedione congeners are highly active inhibitors of HIV-1 with EC(50) values ranging from 0.6 to 2 nM in CEM-SS cells infected with laboratory derived viruses, 11-20 nM in fresh human PBMCs infected with subtype B (HT/92/599) virus, and 2-7 nM in PBMCs infected with the clinical subtype C (ZA/97/003) virus. Combination antiviral assays were performed using the laboratory adapted RF strain of HIV-1 in CEM-SS cells and with a clade B and C low passage clinical isolate in fresh human peripheral mononuclear cells and the compound interactions were analyzed using MacSynergy II. The seven pyrimidinedione compounds resulted in additive to synergistic interactions in combination with entry and fusion inhibitors, nonnucleoside and nucleoside reverse transcriptase inhibitors, and the protease inhibitors. No evidence of antagonistic antiviral activity or synergistic cytotoxicity was detected with the combinations of compounds tested. The dual mechanism of action of the pyrimidinediones resulting in inhibition of both virus entry and reverse transcription suggests excellent potential of these lead pyrimidinediones as candidates for combination therapy with other approved HIV inhibitors of varying mechanism of action. Copyright © 2011. Published by Elsevier B.V.

(PCG) Protein Crystal Growth HIV Reverse Transcriptase

NASA Technical Reports Server (NTRS)

1992-01-01

HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

The Discovery of Reverse Transcriptase.

PubMed

Coffin, John M; Fan, Hung

2016-09-29

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology.

PubMed

Collins, Kathleen; Nilsen, Timothy W

2013-08-01

Current investigation of RNA transcriptomes relies heavily on the use of retroviral reverse transcriptases. It is well known that these enzymes have many limitations because of their intrinsic properties. This commentary highlights the recent biochemical characterization of a new family of reverse transcriptases, those encoded by group II intron retrohoming elements. The novel properties of these enzymes endow them with the potential to revolutionize how we approach RNA analyses.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Evidence for retrovirus infections in green turtles Chelonia mydas from the Hawaiian islands

USGS Publications Warehouse

Casey, R.N.; Quackenbush, S.L.; Work, Thierry M.; Balazs, G.H.; Bowser, P.R.; Casey, J.W.

1997-01-01

Apparently normal Hawaiian green turtles Chelonia mydas and those displaying fibropapillomas were analyzed for infection by retroviruses. Strikingly, all samples were positive for polymerase enhanced reverse transcriptase (PERT) with levels high enough to quantitate by the conventional reverse transcriptase (RT) assay. However, samples of skin, even from asymptomatic turtles, were RT positive, although the levels of enzyme activity in healthy turtles hatched and raised in captivity were much lower than those observed in asymptomatic free-ranging turtles. Turtles with fibropapillomas displayed a broad range of reverse transcriptase activity. Skin and eye fibropapillomas and a heart tumor were further analyzed and shown to have reverse transcriptase activity that banded in a sucrose gradient at 1.17 g ml-1. The reverse transcriptase activity purified from the heart tumor displayed a temperature optimum of 37??C and showed a preference for Mn2+ over Mg2+. Sucrose gradient fractions of this sample displaying elevated reverse transcriptase activity contained primarily retrovitalsized particles with prominent envelope spikes, when negatively stained and examined by electron microscopy. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of gradient-purified virions revealed a conserved profile among 4 independent tumors and showed 7 prominent proteins having molecular weights of 116, 83, 51, 43, 40, 20 and 14 kDa. The data suggest that retroviral infections are widespread in Hawaiian green turtles and a comprehensive investigation is warranted to address the possibility that these agents cause green turtle fibropapillomatosis (GTFP).

Short communication: high prevalence of drug resistance in HIV type 1-infected children born in Honduras and Belize 2001 to 2004.

PubMed

Parham, Leda; de Rivera, Ivette Lorenzana; Murillo, Wendy; Naver, Lars; Largaespada, Natalia; Albert, Jan; Karlsson, Annika C

2011-10-01

Antiretroviral therapy has had a great impact on the prevention of mother-to-child transmission (MTCT) of HIV-1. However, development of drug resistance, which could be subsequently transmitted to the child, is a major concern. In Honduras and Belize the prevalence of drug resistance among HIV-1-infected children remains unknown. A total of 95 dried blood spot samples was obtained from HIV-1-infected, untreated children in Honduras and Belize born during 2001 to 2004, when preventive antiretroviral therapy was often suboptimal and consisted of monotherapy with nevirapine or zidovudine. Partial HIV-1 pol gene sequences were successfully obtained from 66 children (Honduras n=55; Belize n=11). Mutations associated with drug resistance were detected in 13% of the Honduran and 27% of the Belizean children. Most of the mutations detected in Honduras (43%) and all mutations detected in Belize were associated with resistance to nonnucleoside reverse transcriptase inhibitors, which was expected from the wide use of nevirapine to prevent MTCT during the study period. In addition, although several mothers reported that they had not received antiretroviral therapy, mutations associated with resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors were found in Honduras. This suggests prior and unreported use of these drugs, or that these women had been infected with resistant virus. The present study demonstrates, for the first time, the presence of drug resistance-associated mutations in HIV-1-infected Honduran and Belizean children.

An overview of the molecular and epidemiological features of HIV-1 infection in two major cities of Bahia state, Brazil.

PubMed

Amaral, Amanda Gm; Oliveira, Isabele B; Carneiro, Diego C; Alcantara, Luiz Cj; Monteiro-Cunha, Joana P

2017-06-01

The high mutation rate of the human immunodeficiency virus (HIV) has created a public health challenge because the use of antiretroviral drugs can generate selective pressure that drives resistance in these viruses. The aim of this work was to characterise the molecular and epidemiological profile of HIV in Bahia, Brazil. DNA sequences from regions of HIV gag, pol, and env genes were obtained from previous studies performed in this area between 2002 and 2012. Their genotype and drug-resistance mutations were identified using bioinformatics tools. Clinical and epidemiological data were analysed. Among 263 individuals (46.4% male), 97.5% were asymptomatic and 49.1% were receiving treatment. Most of the individuals were 31 to 40 years old (36.9%) and infected through heterosexual contact (40.7%). The predominant genotype was B (68.1%) followed by BF recombinants (18.6%). Among the individuals infected with either F or BF genotypes, 68.4% were women and 76.8% were infected through heterosexual transmission. The prevalence of associated mutations conferring antiretroviral resistance was 14.2%, with 3.8% of all mutations conferring resistance to protease inhibitors, 9.43% to nucleoside reverse transcriptase inhibitors, and 8.5% to non-nucleoside reverse transcriptase inhibitors. Drug resistance was higher in individuals receiving treatment (26.1%) than in the drug-naïve (4.3%) individuals. This study will contribute to the understanding and monitoring of HIV epidemic in this Brazilian region.

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors

PubMed Central

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-01-01

Motivation Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. Results We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. Availability A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm. PMID:21234299

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

PubMed

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-12-12

Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

PubMed

Potapov, Vladimir; Fu, Xiaoqing; Dai, Nan; Corrêa, Ivan R; Tanner, Nathan A; Ong, Jennifer L

2018-06-20

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies.more » These results unveil potential medicinal applications of BvvTI.« less

Drug-induced gynecomastia.

PubMed

Bowman, John D; Kim, Hyunah; Bustamante, Juan J

2012-12-01

Drugs account for about 20% of gynecomastia cases in men. As a number of factors can alter the estrogen:androgen ratio, several pathophysiologic mechanisms are associated with drugs causing this disorder. Antiandrogens, protease inhibitors, and nucleoside reverse transcriptase inhibitors are the most common drug causes of gynecomastia, whereas first-generation antipsychotics, spironolactone, verapamil, and cimetidine are less common causes. Other drugs have been reported rarely as causes. Treatment may involve switching to an alternative agent or may require surgery or irradiation if the causative agent cannot be discontinued. We reviewed the literature on drug-induced gynecomastia and provided another perspective by reviewing data from the United States Food and Drug Administration's Adverse Event Reporting System. Epidemiologic studies are needed to provide a more accurate description of the frequency of drug-induced gynecomastia. © 2012 Pharmacotherapy Publications, Inc.

Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

PubMed

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

2010-06-11

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. (c) 2010 Elsevier Inc. All rights reserved.

Impact of lopinavir/ritonavir use on antiretroviral resistance in recent clinical practice.

PubMed

Lambert-Niclot, Sidonie; Masquelier, Bernard; Cohen Codar, Isabelle; Soulie, Cathia; Delaugerre, Constance; Morand-Joubert, Laurence; Charpentier, Charlotte; Ferre, Virginie; Plantier, Jean-Christophe; Montes, Brigitte; Carret, Sophie; Perrot, Valerie; Peytavin, Gilles; Costagliola, Dominique; Calvez, Vincent; Marcelin, Anne-Geneviève

2012-10-01

This observational study was requested by French health authorities to determine the impact of lopinavir/ritonavir (Kaletra(®)) on antiretroviral resistance in clinical practice. Virological failures of lopinavir/ritonavir and their effects on the resistance to protease inhibitors and reverse transcriptase inhibitors were evaluated in protease inhibitor-experienced patients. Virological failure was defined as an HIV-1 plasma viral load >50 copies/mL after at least 3 months of lopinavir/ritonavir-containing antiretroviral therapy. For all patients, a resistance genotypic test was available at failure and before lopinavir/ritonavir treatment. Data from 72 patients with inclusion criteria were studied. The mean viral load at baseline was 4 log(10) copies/mL (1.6-6.5). Mutations in the protease gene significantly selected between baseline and failure were L10V, K20R, L33F, M36I, I47V, I54V, A71V and I85V (P < 0.05). Patients who had more than seven protease inhibitor mutations at baseline showed a significantly increased risk of occurrence of protease inhibitor mutations. The proportion of viruses susceptible to atazanavir, fosamprenavir and darunavir decreased significantly between baseline and failure (P < 0.05). Among patients with a virus susceptible to atazanavir at day 0, 26% (n = 14) exhibited a virus resistant or possibly resistant at the time of failure. This proportion was 32% (n = 16) for fosamprenavir and 16% (n = 7) for darunavir. A darunavir-based regimen appears to be a sequential option in the case of lopinavir/ritonavir failure. To compare and determine the best treatment sequencing, similar studies should be performed for darunavir/ritonavir and atazanavir/ritonavir.

Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

USGS Publications Warehouse

House, M.L.; Kim, C.H.; Reno, P.W.

1998-01-01

Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

PubMed Central

Wainberg, Mark A.

2012-01-01

The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

Nonnucleoside Reverse-transcriptase Inhibitor- vs Ritonavir-boosted Protease Inhibitor-based Regimens for Initial Treatment of HIV Infection: A Systematic Review and Metaanalysis of Randomized Trials.

PubMed

Borges, Álvaro H; Lundh, Andreas; Tendal, Britta; Bartlett, John A; Clumeck, Nathan; Costagliola, Dominique; Daar, Eric S; Echeverría, Patrícia; Gisslén, Magnus; Huedo-Medina, Tania B; Hughes, Michael D; Huppler Hullsiek, Katherine; Khabo, Paul; Komati, Stephanus; Kumar, Princy; Lockman, Shahin; MacArthur, Rodger D; Maggiolo, Franco; Matteelli, Alberto; Miro, Jose M; Oka, Shinichi; Petoumenos, Kathy; Puls, Rebekah L; Riddler, Sharon A; Sax, Paul E; Sierra-Madero, Juan; Torti, Carlo; Lundgren, Jens D

2016-07-15

Previous studies suggest that nonnucleoside reverse-transcriptase inhibitors (NNRTIs) cause faster virologic suppression, while ritonavir-boosted protease inhibitors (PI/r) recover more CD4 cells. However, individual trials have not been powered to compare clinical outcomes. We searched databases to identify randomized trials that compared NNRTI- vs PI/r-based initial therapy. A metaanalysis calculated risk ratios (RRs) or mean differences (MDs), as appropriate. Primary outcome was death or progression to AIDS. Secondary outcomes were death, progression to AIDS, and treatment discontinuation. We calculated RR of virologic suppression and MD for an increase in CD4 cells at week 48. We included 29 trials with 9047 participants. Death or progression to AIDS occurred in 226 participants in the NNRTI arm and in 221 in the PI/r arm (RR, 1.03; 95% confidence interval, .87-1.22; 12 trials; n = 3825), death in 205 participants in the NNRTI arm vs 198 in the PI/r arm (1.04; 0.86-1.25; 22 trials; n = 8311), and progression to AIDS in 140 participants in the NNRTI arm vs 144 in the PI/r arm (1.00; 0.80-1.25; 13 trials; n = 4740). Overall treatment discontinuation (1.12; 0.93-1.35; 24 trials; n = 8249) and from toxicity (1.21; 0.87-1.68; 21 trials; n = 6195) were comparable, but discontinuation due to virologic failure was more common with NNRTI (1.58; 0.91-2.74; 17 trials; n = 5371). At week 48, there was no difference between NNRTI and PI/r in virologic suppression (RR, 1.03; 0.98-1.09) or CD4(+) recovery (MD, -4.7 cells; -14.2 to 4.8). We found no difference in clinical and viro-immunologic outcomes between NNRTI- and PI/r-based therapy. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

PubMed

Tavallaee, Mahkam; Steiner, David F; Zehnder, James L; Folkins, Ann K; Karam, Amer K

2018-04-03

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.

Elevated Human telomerase reverse transcriptase gene expression in blood cells associated with chronic and arsenic exposure in Inner Mongolia, China

EPA Science Inventory

BACKGROUND: Arsenic exposure is associated with human cancer. Telomerase containing the catalytic subunit, human telomerase reverse transcriptase (hTERT), can extend telomeres of chromosomes, delay senescence and promoting cell proliferation leading to tumorigenesis. OBJECTIVE:...

Boosted protease inhibitor monotherapy in HIV-infected adults: outputs from a pan-European expert panel meeting

PubMed Central

2013-01-01

While the introduction of combination highly active antiretroviral therapy (HAART) regimens represents an important advance in the management of human immunodeficiency virus (HIV)-infected patients, tolerability can be an issue and the use of several different agents may produce problems. The switch of combination HAART to ritonavir-boosted protease inhibitor (PI) monotherapy may offer the opportunity to maintain antiviral efficacy while reducing treatment complexity and the risks of toxicity. Current European AIDS Clinical Society (EACS) guidelines recognise ritonavir-boosted PI monotherapy with twice-daily lopinavir/ritonavir or once-daily darunavir/ritonavir as a possible option in patients who have intolerance to nucleoside reverse transcriptase inhibitors, or for treatment simplification. Clinical trials data for PI boosted monotherapy are encouraging, showing substantial efficacy in the majority of patients; however, further data are required before this approach can be recommended as a routine treatment. Available data indicate that the most suitable candidates for the use of boosted PI monotherapy are long-term virologically suppressed patients who have demonstrated good adherence to antiretroviral therapy, who do not have chronic hepatitis B, have no history of treatment failure on PIs and are able to tolerate low-dose ritonavir. PMID:23347595

Prevalence and response to antiretroviral therapy of non-B subtypes of HIV in antiretroviral-naive individuals in British Columbia.

PubMed

Alexander, Christopher S; Montessori, Valentina; Wynhoven, Brian; Dong, Winnie; Chan, Keith; O'Shaughnessy, Michael V; Mo, Theresa; Piaseczny, Magda; Montaner, Julio S G; Harrigan, P Richard

2002-03-01

In North America, the B subtype of the major group (M) of HIV-1 predominates. Phylogenetic analysis of HIV reverse transcriptase and protease sequences isolated from 479 therapy-naive patients, first seeking treatment in British Columbia between June 1997 and August 1998, revealed a prevalence of 4.4% non-B virus. A range of different subtypes was identified, including one subtype A, 11 C, two D, five CRF01_AE, and one sample that could not be reliably subtyped. Baseline CD4 courts were significantly lower in individuals harbouring the non-B subtypes (P = 0.02), but baseline viral loads were similar (P = 0.80). In this study, individuals infected with non-B variants did not have a significantly different virological response to therapy after up to 18 months.

Initiating therapy: when to start, what to use.

PubMed

Hirsch, Martin S

2008-05-15

Decisions regarding whether to start combination antiretroviral therapy (cART) during primary infection and when to initiate treatment during chronic infection continue to evolve. Although current data suggest that there may be a benefit to therapy during primary infection, results are inconclusive. Once begun, treatment probably should be continued indefinitely, since its potential advantages disappear over time if treatment is stopped. Recent studies suggest that cART may be useful at higher CD4 cell count thresholds than are currently recommended in several guidelines. Several regimens are acceptable as initial therapy, with tenofovir/emtricitabine/efavirenz favored by many because of potency and ease of administration. Other favored regimens include combinations of 2 nucleoside (or nucleotide) reverse-transcriptase inhibitors and a ritonavir-boosted protease inhibitor. Some new antiretroviral drugs under study, particularly integrase inhibitors, may prove useful in treatment-naive patients.

Brief Report: Long-Term (96-Week) Efficacy and Safety After Switching From Tenofovir Disoproxil Fumarate to Tenofovir Alafenamide in HIV-Infected, Virologically Suppressed Adults

PubMed Central

Raffi, François; Orkin, Chloe; Clarke, Amanda; Slama, Laurence; Gallant, Joel; Daar, Eric; Henry, Keith; Santana-Bagur, Jorge; Stein, David K.; Bellos, Nicholaos; Scarsella, Anthony; Yan, Mingjin; Abram, Michael E.; Cheng, Andrew

2017-01-01

Abstract: In a double-blind, phase 3 trial, 663 HIV-infected, virologically suppressed adults were randomized to switch to tenofovir alafenamide (TAF; n = 333) vs. remain on tenofovir disoproxil fumarate (TDF; n = 330), each coformulated with emtricitabine (FTC), while continuing their third agent (boosted protease inhibitor or unboosted third agent). At week 96, 88.6% on FTC/TAF and 89.1% on FTC/TDF had HIV-1 RNA <50 copies per milliliter [adjusted difference −0.5% (95% confidence interval: −5.3 to 4.4%)]. Proteinuria, albuminuria, proximal renal tubular function, and bone mineral density improved after switching to TAF- from TDF-containing regimens. These longer-term data support FTC/TAF as a safe, well-tolerated, and durable nucleotide reverse transcriptase inhibitor backbone. PMID:28272164

Anti-adenoviral effect of anti-HIV agents in vitro in serotypes inducing keratoconjunctivitis.

PubMed

Uchio, Eiichi; Fuchigami, Aki; Kadonosono, Kazuaki; Hayashi, Akio; Ishiko, Hiroaki; Aoki, Koki; Ohno, Shigeaki

2007-09-01

Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC(50) (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

The impact of transmission clusters on primary drug resistance in newly diagnosed HIV-1 infection.

PubMed

Yerly, Sabine; Junier, Thomas; Gayet-Ageron, Angèle; Amari, Emmanuelle Boffi El; von Wyl, Viktor; Günthard, Huldrych F; Hirschel, Bernard; Zdobnov, Evgeny; Kaiser, Laurent

2009-07-17

To monitor HIV-1 transmitted drug resistance (TDR) in a well defined urban area with large access to antiretroviral therapy and to assess the potential source of infection of newly diagnosed HIV individuals. All individuals resident in Geneva, Switzerland, with a newly diagnosed HIV infection between 2000 and 2008 were screened for HIV resistance. An infection was considered as recent when the positive test followed a negative screening test within less than 1 year. Phylogenetic analyses were performed by using the maximum likelihood method on pol sequences including 1058 individuals with chronic infection living in Geneva. Of 637 individuals with newly diagnosed HIV infection, 20% had a recent infection. Mutations associated with resistance to at least one drug class were detected in 8.5% [nucleoside reverse transcriptase inhibitors (NRTIs), 6.3%; non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3.5%; protease inhibitors, 1.9%]. TDR (P-trend = 0.015) and, in particular, NNRTI resistance (P = 0.002) increased from 2000 to 2008. Phylogenetic analyses revealed that 34.9% of newly diagnosed individuals, and 52.7% of those with recent infection were linked to transmission clusters. Clusters were more frequent in individuals with TDR than in those with sensitive strains (59.3 vs. 32.6%, respectively; P < 0.0001). Moreover, 84% of newly diagnosed individuals with TDR were part of clusters composed of only newly diagnosed individuals. Reconstruction of the HIV transmission networks using phylogenetic analysis shows that newly diagnosed HIV infections are a significant source of onward transmission, particularly of resistant strains, thus suggesting an important self-fueling mechanism for TDR.

Executive summary of the GESIDA/National AIDS Plan Consensus Document on antiretroviral therapy in adults infected by the human immunodeficiency virus (updated January 2015).

PubMed

Berenguer, Juan; Polo, Rosa; Aldeguer, José López; Lozano, Fernando; Aguirrebengoa, Koldo; Arribas, José Ramón; Blanco, José Ramón; Boix, Vicente; Casado, José Luis; Clotet, Bonaventura; Crespo, Manuel; Domingo, Pere; Estrada, Vicente; García, Federico; Gatell, José María; González-García, Juan; Gutiérrez, Félix; Iribarren, José Antonio; Knobel, Hernando; Llibre, Josep María; Locutura, Jaime; López, Juan Carlos; Miró, José M; Moreno, Santiago; Podzamczer, Daniel; Portilla, Joaquín; Pulido, Federico; Ribera, Esteban; Riera, Melchor; Rubio, Rafael; Santos, Jesús; Sanz-Moreno, José; Sanz, Jesús; Téllez, María Jesús; Tuset, Montserrat; Rivero, Antonio

2015-10-01

In this update, antiretroviral therapy (ART) is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The strength and grade of the recommendation vary depending on the CD4+ T-lymphocyte count, the presence of opportunistic infections or comorbid conditions, age, and the efforts to prevent the transmission of HIV. The objective of ART is to achieve an undetectable plasma viral load (PVL). Initial ART should comprise three drugs, namely, two nucleoside reverse transcriptase inhibitors (NRTI) and one drug from another family. Three of the recommended regimens, all of which have an integrase strand transfer inhibitor (INSTI) as the third drug, are considered a preferred regimen; a further seven regimens, which are based on an INSTI, an non-nucleoside reverse transcriptase inhibitor (NNRTI), or a protease inhibitor boosted with ritonavir (PI/r), are considered alternatives. The reasons and criteria for switching ART are presented both for patients with an undetectable PVL and for patients who experience virological failure, in which case the rescue regimen should include three (or at least two) drugs that are fully active against HIV. The specific criteria for ART in special situations (acute infection, HIV-2 infection, pregnancy) and comorbid conditions (tuberculosis and other opportunistic infections, kidney disease, liver disease, and cancer) are updated. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Antiretroviral treatment sequencing strategies to overcome HIV type 1 drug resistance in adolescents and adults in low-middle-income countries.

PubMed

De Luca, Andrea; Hamers, Raphael L; Schapiro, Jonathan M

2013-06-15

Antiretroviral treatment (ART) is expanding to human immunodeficiency virus type 1 (HIV-1)-infected persons in low-middle income countries, thanks to a public health approach. With 3 available drug classes, 2 ART sequencing lines are programmatically foreseen. The emergence and transmission of viral drug resistance represents a challenge to the efficacy of ART. Knowledge of HIV-1 drug resistance selection associated with specific drugs and regimens and the consequent activity of residual drug options are essential in programming ART sequencing options aimed at preserving ART efficacy for as long as possible. This article determines optimal ART sequencing options for overcoming HIV-1 drug resistance in resource-limited settings, using currently available drugs and treatment monitoring opportunities. From the perspective of drug resistance and on the basis of limited virologic monitoring data, optimal sequencing seems to involve use of a tenofovir-containing nonnucleoside reverse-transcriptase inhibitor-based first-line regimen, followed by a zidovudine-containing, protease inhibitor (PI)-based second-line regimen. Other options and their consequences are explored by considering within-class and between-class sequencing opportunities, including boosted PI monotherapies and future options with integrase inhibitors. Nucleoside reverse-transcriptase inhibitor resistance pathways in HIV-1 subtype C suggest an additional reason for accelerating stavudine phase out. Viral load monitoring avoids the accumulation of resistance mutations that significantly reduce the activity of next-line options. Rational use of resources, including broader access to viral load monitoring, will help ensure 3 lines of fully active treatment options, thereby increasing the duration of ART success.

Prevalence of antiretroviral drug resistance and resistance-associated mutations in antiretroviral therapy-naïve HIV-infected individuals from 40 United States cities.

PubMed

Ross, Lisa; Lim, Michael L; Liao, Qiming; Wine, Brian; Rodriguez, Allan E; Weinberg, Winkler; Shaefer, Mark

2007-01-01

Transmission of drug-resistant HIV strains to antiretroviral therapy (ART)-naïve subjects can negatively impact therapy response. As treatment strategies and utilization of antiretroviral drugs evolve, patterns of transmitted mutations may shift. Paired genotypic and phenotypic susceptibility data were retrospectively analyzed for 317 ART-naïve, HIV-infected subjects from 40 small and major metropolitan cities in the Northeastern, Midwestern, Southern, Southwestern, and Northwestern United States during 2003. Using current (January 2007) PhenoSense cutoffs, HIV-from 8% of subjects had reduced susceptibility to > or = 1 drug. By class, < 1% had reduced susceptibility to protease inhibitors (PIs), and 1% had reduced susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs); reduced susceptibility to > or = 1 non-nucleoside reverse transcriptase inhibitor (NNRTIs) was seen in 7% of subjects, with 4% of all subjects having reduced susceptibility to all NNRTIs. IAS-USA-defined NRTI, NNRTI, and/or major PI HIV-drug resistance-associated mutations were detected for 0% of the subjects. HIV risk factors included homosexual contact (74%), heterosexual contact (28%), and injectable drug use/transfusion/other (7%). Reduced susceptibility to > or = 1 drug was significantly higher (p = .034) for white subjects than African Americans and Hispanics/others. The high prevalence of drug resistance in these ART-naïve subjects suggests that transmitted resistance is occurring widely within the United States. HIV genotyping and/or phenotyping for antiretroviral-naïve patients seeking treatment should be considered, especially if the therapy will include an NNRTI.

Development and validation of the first liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of multiple antiretrovirals in meconium.

PubMed

Himes, Sarah K; Scheidweiler, Karl B; Tassiopoulos, Katherine; Kacanek, Deborah; Hazra, Rohan; Rich, Kenneth; Huestis, Marilyn A

2013-02-05

A novel method for the simultaneous quantification of 16 antiretroviral (ARV) drugs and 4 metabolites in meconium was developed and validated. Quantification of 6 nucleoside/nucleotide reverse transcriptase inhibitors, 2 non-nucleoside reverse transcriptase inhibitors, 7 protease inhibitors, and 1 integrase inhibitor was achieved in 0.25 g of meconium. Specimen preparation included methanol homogenization and solid-phase extraction. Separate positive and negative polarity multiple reaction monitoring mode injections were required to achieve sufficient sensitivity. Linearity ranged from 10 to 75 ng/g up to 2500 ng/g for most analytes and 100-500 ng/g up to 25,000 ng/g for some; all correlation coefficients were ≥0.99. Extraction efficiencies from meconium were 32.8-119.5% with analytical recovery of 80.3-108.3% and total imprecision of 2.2-11.0% for all quantitative analytes. Two analytes with analytical recovery (70.0-138.5%) falling outside the 80-120% criteria range were considered semiquantitative. Matrix effects were -98.3-47.0% and -98.0-67.2% for analytes and internal standards, respectively. Analytes were stable (>75%) at room temperature for 24 h, 4 °C for 3 days, -20 °C for 3 freeze-thaw cycles over 3 days, and on the autosampler. Method applicability was demonstrated by analyzing meconium from HIV-uninfected infants born to HIV-positive mothers on ARV therapy. This method can be used as a tool to investigate the potential effects of in utero ARV exposure on childhood health and neurodevelopmental outcomes.

Prevalence of Transmitted HIV Drug Resistance Among Recently Infected Persons in San Diego, CA 1996-2013.

PubMed

Panichsillapakit, Theppharit; Smith, Davey M; Wertheim, Joel O; Richman, Douglas D; Little, Susan J; Mehta, Sanjay R

2016-02-01

Transmitted drug resistance (TDR) remains an important concern when initiating antiretroviral therapy (ART). Here, we describe the prevalence and phylogenetic relationships of TDR among ART-naive, HIV-infected individuals in San Diego from 1996 to 2013. Data were analyzed from 496 participants of the San Diego Primary Infection Cohort who underwent genotypic resistance testing before initiating therapy. Mutations associated with drug resistance were identified according to the WHO-2009 surveillance list. Network and phylogenetic analyses of the HIV-1 pol sequences were used to evaluate the relationships of TDR within the context of the entire cohort. The overall prevalence of TDR was 13.5% (67/496), with an increasing trend over the study period (P = 0.005). TDR was predominantly toward nonnucleoside reverse transcriptase inhibitors (NNRTIs) [8.5% (42/496)], also increasing over the study period (P = 0.005). By contrast, TDR to protease inhibitors and nucleos(t)ide reverse transcriptase inhibitors were 4.4% (22/496) and 3.8% (19/496), respectively, and did not vary with time. TDR prevalence did not differ by age, gender, race/ethnicity, or risk factors. Using phylogenetic analysis, we identified 52 transmission clusters, including 8 with at least 2 individuals sharing the same mutation, accounting for 23.8% (16/67) of the individuals with TDR. Between 1996 and 2013, the prevalence of TDR significantly increased among recently infected ART-naive individuals in San Diego. Around one-fourth of TDR occurred within clusters of recently infected individuals. These findings highlight the importance of baseline resistance testing to guide selection of ART and for public health monitoring.

Anti-human immunodeficiency virus (HIV) activities of halogenated gomisin J derivatives, new nonnucleoside inhibitors of HIV type 1 reverse transcriptase.

PubMed

Fujihashi, T; Hara, H; Sakata, T; Mori, K; Higuchi, H; Tanaka, A; Kaji, H; Kaji, A

1995-09-01

Halogenated gomisin J (a derivative of lignan compound), represented by the bromine derivative 1506 [(6R, 7S, S-biar)-4,9-dibromo-3,10-dihydroxy-1,2,11,12-tetramethoxy-6, 7-dimethyl-5,6,7,8- tetrahydrodibenzo[a,c]cyclo-octene], was found to be a potent inhibitor of the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) on MT-4 human T cells (50% effective dose, 0.1 to 0.5 microM). Gomisin J derivatives were active in preventing p24 production from acutely HIV-1-infected H9 cells. The selective indices (toxic dose/effective dose) of these compounds were as high as > 300 in some systems. 1506 was active against 3'-azido-3'-deoxythymidine-resistant HIV-1 and acted synergistically with AZT and 2',3'-ddC. 1506 inhibited HIV-1 reverse transcriptase (RT) in vitro but not HIV-1 protease. From the time-of-addition experiment, 1506 was found to inhibit the early phase of the HIV life cycle. A 1506-resistant HIV mutant was selected and shown to possess a mutation within the RT-coding region (at position 188 [Tyr to Leu]). The mutant RT expressed in Escherichia coli was resistant to 1506 in the in vitro RT assay. Some of the HIV strains resistant to other nonnucleoside HIV-1 RT inhibitors were also resistant to 1506. Comparison of various gomisin J derivatives with gomisin J showed that iodine, bromine, and chlorine in the fourth and ninth positions increased RT inhibitory activity as well as cytoprotective activity.

Reverse transcriptase activity and particles of retroviral density in cultured canine lymphosarcoma supernatants.

PubMed Central

Tomley, F. M.; Armstrong, S. J.; Mahy, B. W.; Owen, L. N.

1983-01-01

Lymphoid tissue from 43 cases of canine lymphosarcoma and from 40 clinically normal dogs have been examined for markers of retrovirus infection. From 69-76% of culture supernatants from lymphosarcomas were shown to contain particles of retroviral density and to possess poly rC-oligo dG templated polymerase (reverse transcriptase) activity compared with 17-24% of culture supernatants from normal canine lymphoid cells. In 6 culture supernatants from cases of lymphosarcoma, high molecular weight 60-70S RNA was detected and shown to be found in association with this particulate reverse transcriptase activity. No such RNA was detected in 6 culture supernatants from normal canine lymphoid cells. PMID:6186265

Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples

EPA Science Inventory

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since o...

Interaction of aurintricarboxylic acid (ATA) with four nucleic acid binding proteins DNase I, RNase A, reverse transcriptase and Taq polymerase

NASA Astrophysics Data System (ADS)

Ghosh, Utpal; Giri, Kalyan; Bhattacharyya, Nitai P.

2009-12-01

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I ( Kd = 9.019 μM)), RNase A ( Kd = 2.33 μM) reverse transcriptase ( Kd = 0.255 μM) and Taq polymerase ( Kd = 81.97 μM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258

The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition.

PubMed

McNeel, Anthony K; Cushman, Robert A; Vallet, Jeffrey L

2013-06-01

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

Abacavir/lamivudine versus tenofovir/emtricitabine in virologically suppressed patients switching from ritonavir-boosted protease inhibitors to raltegravir.

PubMed

Martínez, Esteban; d'Albuquerque, Polyana M; Pérez, Ignacio; Pich, Judit; Gatell, José M

2013-02-01

There are few clinical data on the combination abacavir/lamivudine plus raltegravir. We compared the outcomes of patients from the SPIRAL trial receiving either abacavir/lamivudine or tenofovir/emtricitabine at baseline who had taken at least one dose of either raltegravir or ritonavir-boosted protease inhibitors. For the purpose of this analysis, treatment failure was defined as virological failure (confirmed HIV-1 RNA ≥50 copies/ml) or discontinuation of abacavir/lamivudine or tenofovir/emtricitabine because of adverse events, consent withdrawal, or lost to follow-up. There were 143 (72.59%) patients with tenofovir/emtricitabine and 54 (27.41%) with abacavir/lamivudine. In the raltegravir group, there were three (11.11%) treatment failures with abacavir/lamivudine and eight (10.96%) with tenofovir/emtricitabine (estimated difference 0.15%; 95% CI -17.90 to 11.6). In the ritonavir-boosted protease inhibitor group, there were four (14.81%) treatment failures with abacavir/lamivudine and 12 (17.14%) with tenofovir/emtricitabine (estimated difference -2.33%; 95% CI -16.10 to 16.70). Triglycerides decreased and HDL cholesterol increased through the study more pronouncedly with abacavir/lamivudine than with tenofovir/emtricitabine and differences in the total-to-HDL cholesterol ratio between both combinations of nucleoside reverse transcriptase inhibitors (NRTIs) tended to be higher in the raltegravir group, although differences at 48 weeks were not significant. While no patient discontinued abacavir/lamivudine due to adverse events, four (2.80%) patients (all in the ritonavir-boosted protease inhibitor group) discontinued tenofovir/emtricitabine because of adverse events (p=0.2744). The results of this analysis do not suggest that outcomes of abacavir/lamivudine are worse than those of tenofovir/emtricitabine when combined with raltegravir in virologically suppressed HIV-infected adults.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors

PubMed Central

Bam, Rujuta A.; Willkom, Madeleine; Frey, Christian R.; Tsai, Luong; Stray, Kirsten M.; Yant, Stephen R.; Cihlar, Tomas

2015-01-01

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4+ T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Structure of HIV-1 nonnucleoside reverse transcriptase inhibitors derivatives of N-benzyl-benzimidazole with different substituents in position 4

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-01-01

The constant development of new drugs against HIV-1 is necessary due to global expansion of AIDS and HIV-1 drug resistance. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic drugs in AIDS therapy. The crystal structures of six nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) derivatives of N-benzyl-benzimidazole are reported here. The investigated compounds belong to the group of so called "butterfly like" inhibitors with characteristic two π-electron moieties with an angled orientation. The structural data show the influence of the substituents of the benzimidazole ring on the geometry of the molecule and correlation between the structure of the inhibitor and its biological activity.

Docking and multivariate methods to explore HIV-1 drug-resistance: a comparative analysis

NASA Astrophysics Data System (ADS)

Almerico, Anna Maria; Tutone, Marco; Lauria, Antonino

2008-05-01

In this paper we describe a comparative analysis between multivariate and docking methods in the study of the drug resistance to the reverse transcriptase and the protease inhibitors. In our early papers we developed a simple but efficient method to evaluate the features of compounds that are less likely to trigger resistance or are effective against mutant HIV strains, using the multivariate statistical procedures PCA and DA. In the attempt to create a more solid background for the prediction of susceptibility or resistance, we carried out a comparative analysis between our previous multivariate approach and molecular docking study. The intent of this paper is not only to find further support to the results obtained by the combined use of PCA and DA, but also to evidence the structural features, in terms of molecular descriptors, similarity, and energetic contributions, derived from docking, which can account for the arising of drug-resistance against mutant strains.

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

Murine Leukemia Viruses: Objects and Organisms

PubMed Central

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera. PMID:22312342

Murine leukemia viruses: objects and organisms.

PubMed

Rein, Alan

2011-01-01

Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes-protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.

Antiretroviral Therapy for HIV-2 Infection: Recommendations for Management in Low-Resource Settings

PubMed Central

Peterson, Kevin; Jallow, Sabelle; Rowland-Jones, Sarah L.; de Silva, Thushan I.

2011-01-01

HIV-2 contributes approximately a third to the prevalence of HIV in West Africa and is present in significant amounts in several low-income countries outside of West Africa with historical ties to Portugal. It complicates HIV diagnosis, requiring more expensive and technically demanding testing algorithms. Natural polymorphisms and patterns in the development of resistance to antiretrovirals are reviewed, along with their implications for antiretroviral therapy. Nonnucleoside reverse transcriptase inhibitors, crucial in standard first-line regimens for HIV-1 in many low-income settings, have no effect on HIV-2. Nucleoside analogues alone are not sufficiently potent enough to achieve durable virologic control. Some protease inhibitors, in particular those without ritonavir boosting, are not sufficiently effective against HIV-2. Following review of the available evidence and taking the structure and challenges of antiretroviral care in West Africa into consideration, the authors make recommendations and highlight the needs of special populations. PMID:21490779

Antiretroviral Drug Use in a Cohort of HIV-Uninfected Women in the United States: HIV Prevention Trials Network 064.

PubMed

Chen, Iris; Clarke, William; Ou, San-San; Marzinke, Mark A; Breaud, Autumn; Emel, Lynda M; Wang, Jing; Hughes, James P; Richardson, Paul; Haley, Danielle F; Lucas, Jonathan; Rompalo, Anne; Justman, Jessica E; Hodder, Sally L; Eshleman, Susan H

2015-01-01

Antiretroviral (ARV) drug use was analyzed in HIV-uninfected women in an observational cohort study conducted in 10 urban and periurban communities in the United States with high rates of poverty and HIV infection. Plasma samples collected in 2009-2010 were tested for the presence of 16 ARV drugs. ARV drugs were detected in samples from 39 (2%) of 1,806 participants: 27/181 (15%) in Baltimore, MD and 12/179 (7%) in Bronx, NY. The ARV drugs detected included different combinations of non-nucleoside reverse transcriptase inhibitors and protease inhibitors (1-4 drugs/sample). These data were analyzed in the context of self-reported data on ARV drug use. None of the 39 women who had ARV drugs detected reported ARV drug use at any study visit. Further research is needed to evaluate ARV drug use by HIV-uninfected individuals.

Antiretroviral Drug Use in a Cohort of HIV-Uninfected Women in the United States: HIV Prevention Trials Network 064

PubMed Central

Chen, Iris; Clarke, William; Ou, San-San; Marzinke, Mark A.; Breaud, Autumn; Emel, Lynda M.; Wang, Jing; Hughes, James P.; Richardson, Paul; Haley, Danielle F.; Lucas, Jonathan; Rompalo, Anne; Justman, Jessica E.; Hodder, Sally L.; Eshleman, Susan H.

2015-01-01

Antiretroviral (ARV) drug use was analyzed in HIV-uninfected women in an observational cohort study conducted in 10 urban and periurban communities in the United States with high rates of poverty and HIV infection. Plasma samples collected in 2009–2010 were tested for the presence of 16 ARV drugs. ARV drugs were detected in samples from 39 (2%) of 1,806 participants: 27/181 (15%) in Baltimore, MD and 12/179 (7%) in Bronx, NY. The ARV drugs detected included different combinations of non-nucleoside reverse transcriptase inhibitors and protease inhibitors (1–4 drugs/sample). These data were analyzed in the context of self-reported data on ARV drug use. None of the 39 women who had ARV drugs detected reported ARV drug use at any study visit. Further research is needed to evaluate ARV drug use by HIV-uninfected individuals. PMID:26445283

Drug Interactions and Antiretroviral Drug Monitoring

PubMed Central

Foy, Matthew; Sperati, C. John; Lucas, Gregory M.

2014-01-01

Due to the improved longevity afforded by combination antiretroviral therapy (cART), HIV-infected individuals are developing several non-AIDS related comorbid conditions. Consequently, medical management of the HIV-infected population is increasingly complex, with a growing list of potential drug-drug interactions (DDIs). This article reviews some of the most relevant and emerging potential interactions between antiretroviral medications and other agents. The most common DDIs are those involving protease inhibitors or non-nucleoside reverse transcriptase inhibitors which alter the cytochrome P450 enzyme system and/or drug transporters such as p-glycoprotein. Of note are the new agents for the treatment of chronic hepatitis C virus infection. These new classes of drugs and others drugs which are increasingly used in this patient population represent a significant challenge with regard to achieving the goals of effective HIV suppression and minimization of drug-related toxicities. Awareness of DDIs and a multidisciplinary approach are imperative in reaching these goals. PMID:24950731

Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

PubMed

Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

2016-07-01

Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Telomerase reverse transcriptase (TERT) expression and role of vincristine sulfate in mouse model of malignancy related peritoneal ascites: an experimental metastatic condition.

PubMed

Chaklader, M; Das, P; Pereira, J A; Chatterjee, S; Basak, P; Law, A; Banerjee, T; Chauhan, S; Law, S

2011-06-01

To evaluate the efficacy of intraperitoneal vincristine administration into ascitic sarcoma-180 bearing mice as a model of human malignant ascites regarding various peritoneal/retroperitoneal sarcomatosis, and to evaluate the flowcytometric telomerase reverse transcriptase expression for the diagnostic and prognostic purposes. Present study included disease induction by intraperitoneal homologous ascitic sarcoma-180 transplantation followed by in vivo intraperitoneal drug administration to study mitotic index, flowcytometric cell cycle and telomerase reverse transcriptase expression pattern, erythrosin-B dye exclusion study for malignant cell viability assessment. Besides, in vitro malignant ascite culture in presence and absence of vincristine sulfate and survival study were also taken into consideration. Intraperitoneal vincristine administration (concentration 0.5 mg/kg body weight) significantly diminished the mitotic index in diseased subjects in comparison to untreated control subjects. Treated group of animals showed increased life span and median survival time. Cell viability assessment during the course of drug administration also revealed gradual depression on cell viability over time. Flowcytometric cell cycle analysis showed a good prognostic feature of chemotherapeutic administration schedule by representing high G2/M phase blocked cells along with reduced telomerase reverse transcriptase positive cells in treated animals. We conclude that long term administration of vincristine sulfate in small doses could be a good pharmacological intervention in case of malignant peritoneal ascites due to sarcomatosis as it indirectly reduced the level of telomerase reverse transcriptase expression in malignant cells by directly regulating cell cycle and simultaneously increased the life expectancy of the diseased subjects.

The effect of transmitted HIV-1 drug resistance on pre-therapy viral load.

PubMed

Harrison, Linda; Castro, Hannah; Cane, Patricia; Pillay, Deenan; Booth, Clare; Phillips, Andrew; Geretti, Anna Maria; Dunn, David

2010-07-31

Reduced replication capacity of viruses expressing drug resistant mutations implies that patients with transmitted drug resistance (TDR) could have lower HIV RNA viral load than those infected with wild-type virus. We performed analysis using data from the UK HIV Drug Resistance Database and the UK CHIC study. Eligible patients had a resistance test performed between 1997 and 2007 while naive to antiretroviral therapy, were 16 years or older, and had a viral load and CD4 cell count measurement within 6 months of this test. Models were adjusted for CD4 cell count, viral subtype, ethnicity, risk group, sex, age, calendar year, clinical centre, and viral load assay. Of a total of 7994 patients included, 709 (9%) had TDR: 604 (85%) had resistance to one drug class only [350 nucleos(t)ide reverse transcriptase inhibitors (NRTIs), 164 non-nucleos(t)ide reverse transcriptase inhibitors (NNRTIs), 90 protease inhibitors (PIs)], 77 (11%) to two classes (42 NRTIs/NNRTIs, 31 NRTIs/PIs, 4 NNRTIs/PIs), and 28 (4%) had resistance to all three classes. The overall mean (SD) viral load at the time of resistance testing was 4.60 (0.82) log(10) copies/ml, and did not differ by class of TDR. However, patients harbouring M184V/I (n = 61) had a significantly lower viral load [adjusted mean difference -0.33 log10 copies/ml (95% CI -0.54 to -0.11), 53% lower (95% CI 22 to 71%), P = 0.002] compared to wild-type virus. Our study provides clear evidence of an in-vivo fitness cost associated with the M184V/I mutation independent of drug effects which select for this mutation. This was not observed for any other mutation, but true effects may have been obscured by reversion of initially resistant viruses to wild-type.

Exposure to antiretroviral therapy and risk of cancer in HIV-infected persons.

PubMed

Chao, Chun; Leyden, Wendy A; Xu, Lanfang; Horberg, Michael A; Klein, Daniel; Towner, William J; Quesenberry, Charles P; Abrams, Donald I; Silverberg, Michael J

2012-11-13

The incidence of certain non-AIDS-defining cancers (NADCs) in HIV patients has been reported to have increased in the combination antiretroviral therapy (ART) era. Studies are needed to directly evaluate the effect of ART use on cancer risk. We followed 12 872 HIV-infected Kaiser Permanente members whose complete ART history was known for incident cancers between 1996 and 2008. Cancers, identified from Surveillance, Epidemiology, and End Results (SEER)-based cancer registries, were grouped as ADCs, infection-related NADCs, or infection-unrelated NADCs. We also evaluated the most common individual cancer types. Rate ratios for ART use (yes/no) and cumulative duration of any ART, protease inhibitor, and nonnucleotide reverse transcriptase inhibitor (NNRTI) therapy were obtained from Poisson models adjusting for demographics, pretreatment or recent CD4 cell count and HIV RNA levels, years known HIV-infected, prior antiretroviral use, HIV risk, smoking, alcohol/drug abuse, overweight/obesity, and calendar year. The cohort experienced 32 368 person-years of ART, 21 249 person-years of protease inhibitor therapy, and 15 643 person-years of NNRTI therapy. The mean follow-up duration was 4.5 years. ADC rates decrease with increased duration of ART use [rate ratio per year = 0.61 (95% confidence interval 0.56-0.66)]; the effect was similar by therapy class. ART, protease inhibitor, or NNRTI therapy duration was not associated with infection-related or infection-unrelated NADC [rate ratio per year ART = 1.00 (0.91-1.11) and 0.96 (0.90-1.01), respectively], except a higher anal cancer risk with longer protease inhibitor therapy [rate ratio per year = 1.16 (1.02-1.31)]. No therapy class-specific effect was found for ADC. ART exposure was generally not associated with NADC risk, except for long-term use of protease inhibitor, which might be associated with increased anal cancer risk.

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

PubMed

Blanca, Giuseppina; Baldanti, Fausto; Paolucci, Stefania; Skoblov, Alexander Yu; Victorova, Lyubov; Hübscher, Ulrich; Gerna, Giuseppe; Spadari, Silvio; Maga, Giovanni

2003-05-02

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

EPA Science Inventory

A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

PubMed

Shulman, Nancy S; Delgado, Jamael; Bosch, Ronald J; Winters, Mark A; Johnston, Elizabeth; Shafer, Robert W; Katzenstein, David A; Merigan, Thomas C

2005-05-01

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Discovery of piperidin-4-yl-aminopyrimidine derivatives as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Wan, Zheng-Yong; Yao, Jin; Tao, Yuan; Mao, Tian-Qi; Wang, Xin-Long; Lu, Yi-Pei; Wang, Hai-Feng; Yin, Hong; Wu, Yan; Chen, Fen-Er; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

2015-06-05

A novel series of piperidin-4-yl-aminopyrimidine derivatives were designed fusing the pharmacophore templates of etravirine-VRX-480773 hybrids our group previously described and piperidine-linked aminopyrimidines. Most compounds displayed significantly improved activity against wild-type HIV-1 with EC50 values in single-digit nanomolar concentrations compared to etravirine-VRX-480773 hybrids. Selected compounds were also evaluated for activity against reverse transcriptase, and had lower IC50 values than that of nevirapine. The improved potency observed in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits reverse transcriptase, and represents a remarkable step forward in the development of AIDS therapeutics. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Transmitted drug resistance and type of infection in newly diagnosed HIV-1 individuals in Honduras.

PubMed

Murillo, Wendy; Paz-Bailey, Gabriela; Morales, Sonia; Monterroso, Edgar; Paredes, Mayte; Dobbs, Trudy; Parekh, Bharat S; Albert, Jan; Rivera, Ivette Lorenzana de

2010-12-01

Transmitted drug resistance (TDR) reduces the efficacy of antiretroviral treatment and is a public health concern. To gain insight in the epidemiology of TDR in Honduras by evaluating the amount of TDR in a representative sample of newly diagnosed individuals and by determining whether these are recent or established infections. Two hundred treatment-naïve, newly diagnosed HIV-positive individuals representing different population groups (general population, Garifunas ethnic group, female sex workers and men who have sex with men) and different geographic regions were enrolled during April 2004-April 2007. The HIV-1 pol gene was sequenced to identify drug-resistant mutations and TDR was scored as recommended by the WHO. Specimens were classified as recent or established infections using the BED assay. Among 200 samples analyzed from Honduran patients the prevalence of TDR was 7% (95% CI: 3.9-11.5%), 5% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3% for nucleoside reverse transcriptase inhibitors (NRTIs) and 0.5% for protease inhibitors (PIs). Testing of these samples with the BED assay revealed that 12% of the specimens were associated with recent infections. TDR was significantly more common in specimens with recent infection (21%) than established infection (5%) (p=0.016). The prevalence of TDR in Honduras was moderate (7%). The percentage of specimens who were recently infected was low (12%), suggesting that late HIV diagnosis is common. The TDR prevalence was higher in recent than in established infections, which may indicate that TDR is increasing over time. The higher prevalence of NNRTI and NRTI mutations as compared to PI mutations is probably due to a broader and longer use of these drugs in Honduras. Copyright © 2010 Elsevier B.V. All rights reserved.

Efficacy and safety of etravirine (TMC125) in patients with highly resistant HIV-1: primary 24-week analysis.

PubMed

Nadler, Jeffrey P; Berger, Daniel S; Blick, Gary; Cimoch, Paul J; Cohen, Calvin J; Greenberg, Richard N; Hicks, Charles B; Hoetelmans, Richard M W; Iveson, Kathy J; Jayaweera, Dushyantha S; Mills, Anthony M; Peeters, Monika P; Ruane, Peter J; Shalit, Peter; Schrader, Shannon R; Smith, Stephen M; Steinhart, Corklin R; Thompson, Melanie; Vingerhoets, Johan H; Voorspoels, Ellen; Ward, Douglas; Woodfall, Brian

2007-03-30

TMC125-C223 is an open-label, partially blinded, randomized clinical trial to evaluate the efficacy and safety of two dosages of etravirine (TMC125), a non-nucleoside reverse transcriptase inhibitor (NNRTI) with activity against wild-type and NNRTI-resistant HIV-1. A total of 199 patients were randomly assigned 2: 2: 1 to twice-daily etravirine 400 mg, 800 mg and control groups, respectively. The primary endpoint was a change in viral load from baseline at week 24 in the intention-to-treat population. Patients had HIV-1 with genotypic resistance to approved NNRTIs and at least three primary protease inhibitor (PI) mutations. Etravirine groups received an optimized background of at least two approved antiretroviral agents [nucleoside reverse transcriptase inhibitors (NRTI) and/or lopinavir/ritonavir and/or enfuvirtide]. Control patients received optimized regimens of at least three antiretroviral agents (NRTIs or PIs and/or enfuvirtide). The mean change from baseline in HIV-1 RNA at week 24 was -1.04, -1.18 and -0.19 log10 copies/ml for etravirine 400 mg twice a day, 800 mg twice a day and the control group, respectively (P < 0.05 for both etravirine groups versus control). Etravirine showed no dose-related effects on safety and tolerability. No consistent pattern of neuropsychiatric symptoms was observed. There were few hepatic adverse events, and rashes were predominantly early onset and mild to moderate in severity. Etravirine plus an optimized background significantly reduced HIV-1-RNA levels from baseline after 24 weeks in patients with substantial NNRTI and PI resistance, and demonstrated a favorable safety profile compared with control.

Safety of Perinatal Exposure to Antiretroviral Medications: Developmental Outcomes in Infants

PubMed Central

Sirois, Patricia A.; Huo, Yanling; Williams, Paige L.; Malee, Kathleen; Garvie, Patricia A.; Kammerer, Betsy; Rich, Kenneth; Van Dyke, Russell B.; Nozyce, Molly L.

2013-01-01

Background This study evaluated effects of perinatal exposure to antiretroviral (ARV) medications on neurodevelopment of HIV-exposed, uninfected infants. Methods HIV-exposed, uninfected infants (age 9-15 months) enrolled in SMARTT, a multisite prospective surveillance study, completed the Bayley Scales of Infant and Toddler Development—Third Edition (Bayley-III), assessing cognition, language, motor skills, social-emotional development, and adaptive behavior. Linear regression models were used to evaluate associations between Bayley-III outcomes in infants with and without perinatal and neonatal ARV exposure, by regimen (combination ARV [cARV] versus non-cARV), type of regimen (defined by drug class), and individual ARVs (for infants with cARV exposure), adjusting for maternal and infant health and demographic covariates. Results As of May 2010, 374 infants had valid Bayley-III evaluations. Median age at testing was 12.7 months; 49% male, 79% black, 16% Hispanic. Seventy-nine percent were exposed to regimens containing protease inhibitors (PIs; 9% of PI-containing regimens also included non-nucleoside reverse transcriptase inhibitors [NNRTIs]), 5% to regimens containing NNRTIs (without PI), and 14% to regimens containing only nucleoside reverse transcriptase inhibitors (NRTIs). Overall, 83% were exposed to cARV. No Bayley-III outcome was significantly associated with overall exposure to cARV, ARV regimen, or neonatal prophylaxis. For individual ARVs, following sensitivity analyses, the adjusted group mean on the Language domain was within age expectations but significantly lower for infants with perinatal exposure to atazanavir (p=0.01). Conclusions These results support the safety of perinatal ARV use. Continued monitoring for adverse neurodevelopmental outcomes in older children is warranted, and the safety of atazanavir merits further study. PMID:23340561

Lipoprotein Changes in HIV-Infected Antiretroviral-Naïve Individuals after Starting Antiretroviral Therapy: ACTG Study A5152s Stein: Lipoprotein Changes on Antiretroviral Therapy

PubMed Central

Stein, James H.; Komarow, Lauren; Cotter, Bruno R.; Currier, Judith S.; Dubé, Michael P.; Fichtenbaum, Carl J.; Gerschenson, Mariana; Mitchell, Carol K.C.; Murphy, Robert L.; Squires, Kathleen; Parker, Robert A.; Torriani, Francesca J.

2008-01-01

Background Dyslipidemia is a frequent complication of antiretroviral therapy (ART) for patients with human immunodeficiency virus infection (HIV). The effects of ART on lipoproteins are less well-understood, and have not been investigated in a prospective study where assignment to ART is randomized. Objective To evaluate the effects of three class-sparing ART regimens on lipids and lipoproteins. Methods This was a substudy of a prospective, multicenter study treatment-naïve HIV-infected individuals randomly assigned to receive a regimen of nucleoside reverse transcriptase inhibitors (NRTIs) + the non-nucleoside reverse transcriptase inhibitor efavirenz, NRTIs + the protease inhibitor lopinavir/ritonavir, or a NRTI-sparing regimen of efavirenz + lopinavir/ritonavir. Lipoproteins were measured by nuclear magnetic resonance spectroscopy. Results Among the 82 participants, total and small low-density lipoprotein concentrations increased (median, interquartile range) by 152 (-49 - +407, p<0.01) and 130 (-98 - +417, p<0.01) nmol/L, respectively, especially in the arms containing lopinavir/ritonavir (pKW<0.04). Very low-density lipoproteins also increased (p<0.01), with a larger increase in the arms that contained lopinavir/ritonavir (p=0.022). High-density lipoproteins increased by 6.0 nmol/L (2.8 - 10.4, p<0.01), but differences between arms were not significant (pKW=0.069). Changes were not related to changes in markers of insulin/glucose metabolism. Conclusions Total and small low-density lipoprotein concentrations increased, especially in the arms containing lopinavir/ritonavir, as did increases in total very low-density lipoproteins. Adverse changes were especially prominent in the arm with efavirenz + lopinavir/ritonavir. PMID:19956354

First trimester maternal uterine artery Doppler examination in HIV-positive women.

PubMed

Savvidou, M D; Samuel, M I; Akolekar, R; Poulton, M; Nicolaides, K H

2011-11-01

The aim of the current study was to assess the effect of maternal HIV infection, treated or untreated, on the degree of placental invasion, as assessed by the pulsatility index of the uterine arteries during a Doppler examination at 11(+0) -13(+6) weeks' gestation. This was a nested case-control study in which a uterine artery Doppler examination was performed in the first trimester in 76 HIV-positive women. Each woman was matched with 30 HIV-negative women. As the pulsatility index of the uterine arteries depends on a number of maternal and fetal characteristics, its values in each case and control were expressed as multiples of the median (MoM) of the unaffected group. Among the 76 HIV-positive women, 33 (43.4%) were on antiretroviral treatment at the time of the Doppler examination, including 14 women (42.4%) on nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor, 18 women (54.5%) on NRTIs and a nonnucleoside reverse transcriptase inhibitor and one woman (3.1%) on monotherapy. Compared with the HIV-negative women, the HIV-positive women were more likely to be heavier (P<0.01), to be of African origin (P<0.01), to be nonsmokers (P=0.01) and to deliver smaller neonates earlier (P<0.01). The median adjusted pulsatility index of the uterine arteries was not statistically different between the cases and controls [1.07; interquartile range (IQR) 0.85-1.24 MoM vs. 0.99; IQR 0.81-1.20 MoM; P= 0.28] or, in HIV-positive women, between those receiving and not receiving antiretroviral treatment (P=0.12). HIV-positive women with uncomplicated pregnancies have normal placental perfusion in the first trimester of pregnancy. 2011 British HIV Association.

Prevalence of drug resistance and importance of viral load measurements in Honduran HIV-infected patients failing antiretroviral treatment.

PubMed

Murillo, Wendy; de Rivera, I L; Parham, L; Jovel, E; Palou, E; Karlsson, A C; Albert, J

2010-02-01

The Honduran HIV/AIDS Program began to scale up access to HIV therapy in 2002. Up to May 2008, more than 6000 patients received combination antiretroviral therapy (cART). As HIV drug resistance is the major obstacle for effective treatment, the purpose of this study was to assess the prevalence of antiretroviral drug resistance in Honduran HIV-1-infected individuals. We collected samples from 138 individuals (97 adults and 41 children) on cART with virological, immunological or clinical signs of treatment failure. HIV-1 pol sequences were obtained using an in-house method. Resistance mutations were identified according to the 2007 International AIDS Society (IAS)-USA list and predicted susceptibility to cART was scored using the ANRS algorithm. Resistance mutations were detected in 112 patients (81%), 74% in adults and 98% in children. Triple-, dual- and single-class drug resistance was documented in 27%, 43% and 11% of the study subjects, respectively. Multiple logistic regression showed that resistance was independently associated with type of treatment failure [virological failure (odds ratio (OR) = 1) vs. immunological failure (OR = 0.11; 95% confidence interval (CI) 0.030-0.43) vs. clinical failure (OR = 0.037; 95% CI 0.0063-0.22)], route of transmission (OR = 42.8; 95% CI 3.73-491), and years on therapy (OR = 1.81; 95% CI 1.11-2.93). The prevalence of antiretroviral resistance was high in Honduran HIV-infected patients with signs of treatment failure. A majority of study subjects showed dual- or triple-class resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Virologically defined treatment failure was a strong predictor of resistance, indicating that viral load testing is needed to correctly identify patients with treatment failure attributable to resistance.

Prevalence of HIV drug resistance mutation in the northern Indian population after failure of the first line antiretroviral therapy.

PubMed

Sinha, S; Shekhar, R C; Ahmad, H; Kumar, N; Samantaray, J C; Sreenivas, V; Khan, N H; Mitsuyasu, R T

2012-09-01

There is limited information available about the prevalence and pattern of human immunodeficiency virus (HIV) drug resistance mutations (DRMs) among antiretroviral therapy (ART) experienced patients from northern India. Results of genotypic drug resistance testing were obtained from plasma samples of 128 patients, who had presented with clinical or immunological failure to treatment after at least six months of ART. Major DRMs associated with any of the three classes of antiretroviral (ARV) drugs, nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI), were seen in 120 out of 128 patients (93.8% prevalence). NRTI and NNRTI DRMs were each seen in 115/128 (89.8%) patients, with M184V, M41L, D67N and T215Y being the most frequent among NRTI associated mutations, and K103N, G190A, Y181C and A98G among NNRTI associated ones. PI DRMs were observed in 14/128 (10.9%) patients, with L10I, V82A and L89V being the commonest. These results present a high prevalence of DRMs among ART experienced patients from northern India with clinical or immunological failure of therapy. It emphasizes the need for regular testing of plasma samples of such patients for DRMs in order to detect and replace a failing regimen early, and also the use of HIV drug resistance genotyping of ART naive individuals prior to initiating first line ART for possible transmitted resistance. It is very important to enhance the access of patients to ARV drugs so that their compliance could be improved and hence development of DRMs be minimized.

Selection Pressure in CD8+ T-cell Epitopes in the pol Gene of HIV-1 Infected Individuals in Colombia. A Bioinformatic Approach

PubMed Central

Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J.

2015-01-01

One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells. PMID:25803098

Antiretroviral drug use in a cross-sectional population survey in Africa: NIMH Project Accept (HPTN 043)

PubMed Central

Fogel, Jessica M.; Clarke, William; Kulich, Michal; Piwowar-Manning, Estelle; Breaud, Autumn; Olson, Matthew T.; Marzinke, Mark A.; Laeyendecker, Oliver; Fiamma, Agnès; Donnell, Deborah; Mbwambo, Jessie K. K.; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J.; Eshleman, Susan H.

2016-01-01

Background Antiretroviral (ARV) drug treatment benefits the treated individual and can prevent HIV transmission. We assessed ARV drug use in a community-randomized trial that evaluated the impact of behavioral interventions on HIV incidence. Methods Samples were collected in a cross-sectional survey after a 3-year intervention period. ARV drug testing was performed using samples from HIV-infected adults at four study sites (Zimbabwe; Tanzania; KwaZulu-Natal and Soweto, South Africa; survey period 2009–2011), using an assay that detects 20 ARV drugs (6 nucleoside/nucleotide reverse transcriptase inhibitors [NRTIs]; 3 non-nucleoside reverse transcriptase inhibitors [NNRTIs]; 9 protease inhibitors; maraviroc; raltegravir). Results ARV drugs were detected in 2,011 (27.4%) of 7,347 samples; 88.1% had 1 NNRTI +/− 1–2 NRTIs. ARV drug detection was associated with sex (women>men), pregnancy, older age (>24 years), and study site (p<0.0001 for all four variables). ARV drugs were also more frequently detected in adults who were widowed (p=0.006) or unemployed (p=0.02). ARV drug use was more frequent in intervention versus control communities early in the survey (p=0.01), with a significant increase in control (p=0.004) but not in intervention communities during the survey period. In KwaZulu-Natal, a 1% increase in ARV drug use was associated with a 0.14% absolute decrease in HIV incidence (p=0.018). Conclusions This study used an objective, biomedical approach to assess ARV drug use on a population level. This analysis identified factors associated with ARV drug use and provided information on ARV drug use over time. ARV drug use was associated with lower HIV incidence at one study site. PMID:27828875

Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus.

PubMed Central

Maurer, B; Bannert, H; Darai, G; Flügel, R M

1988-01-01

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae. Images PMID:2451755

Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine

PubMed Central

García-Pérez, Javier; García, Felipe; Blanco, Julia; Escribà-García, Laura; Gatell, Jose Maria; Alcamí, Jose; Plana, Montserrat; Sánchez-Palomino, Sonsoles

2012-01-01

Background The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. Results Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. Conclusions We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles. PMID:23144996

Prevalence and patterns of HIV transmitted drug resistance in Guatemala.

PubMed

Avila-Ríos, Santiago; Mejía-Villatoro, Carlos R; García-Morales, Claudia; Soto-Nava, Maribel; Escobar, Ingrid; Mendizabal, Ricardo; Girón, Amalia; García, Leticia; Reyes-Terán, Gustavo

2011-12-01

To assess human immunodeficiency virus (HIV) diversity and the prevalence of transmitted drug resistance (TDR) in Guatemala. One hundred forty-five antiretroviral treatment-naïve patients referred to the Roosevelt Hospital in Guatemala City were enrolled from October 2010 to March 2011. Plasma HIV pol sequences were obtained and TDR was assessed with the Stanford algorithm and the World Health Organization (WHO) TDR surveillance mutation list. HIV subtype B was highly prevalent in Guatemala (96.6%, 140/145), and a 2.8% (4/145) prevalence of BF1 recombinants and 0.7% (1/145) prevalence of subtype C viruses were found. TDR prevalence for the study period was 8.3% (12/145) with the Stanford database algorithm (score > 15) and the WHO TDR surveillance mutation list. Most TDR cases were associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (83.3%, 10/12); a low prevalence of nucleoside reverse transcriptase inhibitors and protease inhibitors was observed in the cohort (< 1% for both families). Low selection of antiretroviral drug resistance mutations was found, except for NNRTI-associated mutations. Major NNRTI mutations such as K101E, K103N, and E138K showed higher frequencies than expected in ART-naïve populations. Higher literacy was associated with a greater risk of TDR (odds ratio 4.14, P = 0.0264). This study represents one of the first efforts to describe HIV diversity and TDR prevalence and trends in Guatemala. TDR prevalence in Guatemala was at the intermediate level. Most TDR cases were associated with NNRTIs. Further and continuous TDR surveillance is necessary to gain more indepth knowledge about TDR spread and trends in Guatemala and to optimize treatment outcomes in the country.

New Subtypes and Genetic Recombination in HIV Type 1-Infecting Patients with Highly Active Antiretroviral Therapy in Peru (2008–2010)

PubMed Central

Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-01-01

Abstract HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country. PMID:22559065

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

PubMed

Hofstra, L Marije; Sauvageot, Nicolas; Albert, Jan; Alexiev, Ivailo; Garcia, Federico; Struck, Daniel; Van de Vijver, David A M C; Åsjö, Birgitta; Beshkov, Danail; Coughlan, Suzie; Descamps, Diane; Griskevicius, Algirdas; Hamouda, Osamah; Horban, Andrzej; Van Kasteren, Marjo; Kolupajeva, Tatjana; Kostrikis, Leondios G; Liitsola, Kirsi; Linka, Marek; Mor, Orna; Nielsen, Claus; Otelea, Dan; Paraskevis, Dimitrios; Paredes, Roger; Poljak, Mario; Puchhammer-Stöckl, Elisabeth; Sönnerborg, Anders; Staneková, Danica; Stanojevic, Maja; Van Laethem, Kristel; Zazzi, Maurizio; Zidovec Lepej, Snjezana; Boucher, Charles A B; Schmit, Jean-Claude; Wensing, Annemarie M J; Puchhammer-Stockl, E; Sarcletti, M; Schmied, B; Geit, M; Balluch, G; Vandamme, A-M; Vercauteren, J; Derdelinckx, I; Sasse, A; Bogaert, M; Ceunen, H; De Roo, A; De Wit, S; Echahidi, F; Fransen, K; Goffard, J-C; Goubau, P; Goudeseune, E; Yombi, J-C; Lacor, P; Liesnard, C; Moutschen, M; Pierard, D; Rens, R; Schrooten, Y; Vaira, D; Vandekerckhove, L P R; Van den Heuvel, A; Van Der Gucht, B; Van Ranst, M; Van Wijngaerden, E; Vandercam, B; Vekemans, M; Verhofstede, C; Clumeck, N; Van Laethem, K; Beshkov, D; Alexiev, I; Lepej, S Zidovec; Begovac, J; Kostrikis, L; Demetriades, I; Kousiappa, I; Demetriou, V; Hezka, J; Linka, M; Maly, M; Machala, L; Nielsen, C; Jørgensen, L B; Gerstoft, J; Mathiesen, L; Pedersen, C; Nielsen, H; Laursen, A; Kvinesdal, B; Liitsola, K; Ristola, M; Suni, J; Sutinen, J; Descamps, D; Assoumou, L; Castor, G; Grude, M; Flandre, P; Storto, A; Hamouda, O; Kücherer, C; Berg, T; Braun, P; Poggensee, G; Däumer, M; Eberle, J; Heiken, H; Kaiser, R; Knechten, H; Korn, K; Müller, H; Neifer, S; Schmidt, B; Walter, H; Gunsenheimer-Bartmeyer, B; Harrer, T; Paraskevis, D; Hatzakis, A; Zavitsanou, A; Vassilakis, A; Lazanas, M; Chini, M; Lioni, A; Sakka, V; Kourkounti, S; Paparizos, V; Antoniadou, A; Papadopoulos, A; Poulakou, G; Katsarolis, I; Protopapas, K; Chryssos, G; Drimis, S; Gargalianos, P; Xylomenos, G; Lourida, G; Psichogiou, M; Daikos, G L; Sipsas, N V; Kontos, A; Gamaletsou, M N; Koratzanis, G; Sambatakou, H; Mariolis, H; Skoutelis, A; Papastamopoulos, V; Georgiou, O; Panagopoulos, P; Maltezos, E; Coughlan, S; De Gascun, C; Byrne, C; Duffy, M; Bergin, C; Reidy, D; Farrell, G; Lambert, J; O'Connor, E; Rochford, A; Low, J; Coakely, P; O'Dea, S; Hall, W; Mor, O; Levi, I; Chemtob, D; Grossman, Z; Zazzi, M; de Luca, A; Balotta, C; Riva, C; Mussini, C; Caramma, I; Capetti, A; Colombo, M C; Rossi, C; Prati, F; Tramuto, F; Vitale, F; Ciccozzi, M; Angarano, G; Rezza, G; Kolupajeva, T; Vasins, O; Griskevicius, A; Lipnickiene, V; Schmit, J C; Struck, D; Sauvageot, N; Hemmer, R; Arendt, V; Michaux, C; Staub, T; Sequin-Devaux, C; Wensing, A M J; Boucher, C A B; van de Vijver, D A M C; van Kessel, A; van Bentum, P H M; Brinkman, K; Connell, B J; van der Ende, M E; Hoepelman, I M; van Kasteren, M; Kuipers, M; Langebeek, N; Richter, C; Santegoets, R M W J; Schrijnders-Gudde, L; Schuurman, R; van de Ven, B J M; Åsjö, B; Kran, A-M Bakken; Ormaasen, V; Aavitsland, P; Horban, A; Stanczak, J J; Stanczak, G P; Firlag-Burkacka, E; Wiercinska-Drapalo, A; Jablonowska, E; Maolepsza, E; Leszczyszyn-Pynka, M; Szata, W; Camacho, R; Palma, C; Borges, F; Paixão, T; Duque, V; Araújo, F; Otelea, D; Paraschiv, S; Tudor, A M; Cernat, R; Chiriac, C; Dumitrescu, F; Prisecariu, L J; Stanojevic, M; Jevtovic, Dj; Salemovic, D; Stanekova, D; Habekova, M; Chabadová, Z; Drobkova, T; Bukovinova, P; Shunnar, A; Truska, P; Poljak, M; Lunar, M; Babic, D; Tomazic, J; Vidmar, L; Vovko, T; Karner, P; Garcia, F; Paredes, R; Monge, S; Moreno, S; Del Amo, J; Asensi, V; Sirvent, J L; de Mendoza, C; Delgado, R; Gutiérrez, F; Berenguer, J; Garcia-Bujalance, S; Stella, N; de Los Santos, I; Blanco, J R; Dalmau, D; Rivero, M; Segura, F; Elías, M J Pérez; Alvarez, M; Chueca, N; Rodríguez-Martín, C; Vidal, C; Palomares, J C; Viciana, I; Viciana, P; Cordoba, J; Aguilera, A; Domingo, P; Galindo, M J; Miralles, C; Del Pozo, M A; Ribera, E; Iribarren, J A; Ruiz, L; de la Torre, J; Vidal, F; Clotet, B; Albert, J; Heidarian, A; Aperia-Peipke, K; Axelsson, M; Mild, M; Karlsson, A; Sönnerborg, A; Thalme, A; Navér, L; Bratt, G; Karlsson, A; Blaxhult, A; Gisslén, M; Svennerholm, B; Bergbrant, I; Björkman, P; Säll, C; Mellgren, Å; Lindholm, A; Kuylenstierna, N; Montelius, R; Azimi, F; Johansson, B; Carlsson, M; Johansson, E; Ljungberg, B; Ekvall, H; Strand, A; Mäkitalo, S; Öberg, S; Holmblad, P; Höfer, M; Holmberg, H; Josefson, P; Ryding, U

2016-03-01

Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

Effect of dolutegravir in combination with Nucleoside Reverse Transcriptase Inhibitors (NRTIs) on people living with HIV who have pre-existing NRTI mutations.

PubMed

Sörstedt, Erik; Carlander, Christina; Flamholc, Leo; Hejdeman, Bo; Svedhem, Veronica; Sönnerborg, Anders; Gisslén, Magnus; Yilmaz, Aylin

2018-05-01

Until the introduction of dolutegravir (DTG), people living with HIV (PLWH) who have developed nucleoside reverse transcriptase inhibitor (NRTI) mutations have had few other treatment options outside of regimens based on ritonavir-boosted protease inhibitors (PI/r). Here we report treatment results among PLWH in Sweden with pre-existing NRTI mutations on antiretroviral treatment (ART) with DTG and one to two NRTIs. All PLWH on ART with DTG and one to two NRTIs with pre-existing NRTI mutations were retrospectively identified from the National InfCare HIV database. As controls, PLWH on PI/r and one to two NRTIs, matched according to Genotypic Susceptibility Score and observation time, were included. Data were collected as long as the study population was on treatment with DTG; controls were monitored for the same interval. Outcome was classified as either treatment success or failure. In total, 244 participants (122 individuals treated with DTG and 122 individuals treated with PI/r) were included. Median observation time was 78 weeks (interquartile range 50-98 weeks) for participants on DTG and 75 weeks (50-101 weeks) for individuals on PI/r. Viral failure was detected in four individuals treated with DTG and three individuals treated with PI/r, resulting in similar success rates of 96.7% and 97.5%, respectively. No new mutations were found among participants with treatment failure. DTG in combination with one to two NRTIs was as efficient as PI/r in individuals with pre-existing NRTI mutations in this setting. It may be considered an alternative to PI/r-based ART even in the presence of NRTI resistance. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

[Recommendations for non-occupational postexposure HIV prophylaxis. Spanish Working Group on Non-Occupational Postexposure HIV Prophylaxis of the Catalonian Center for Epidemiological Studies on AIDS and the AIDS Study Group].

PubMed

Almeda, Jesús; Casabona, Jordi; Allepuz, Alejandro; García-Alcaide, Felipe; del Romero, Jorge; Tural, Cristina; Colm, Joan; Bolao, Ferrán; Campins, Magda; Domínguez, Angela; Force, Lluís; Giménez, Albert; Guerra-Romero, Luis

2002-10-01

Evidence is lacking on the possible efficacy and effectiveness of non-occupational postexposure prophylaxis (PEP). However, because of its biological plausibility, the use of antiretroviral (ARV) drugs to prevent the development of infection in certain cases of accidental or sporadic exposure has begun to be considered as common clinical practice. Previous studies performed in Spain have demonstrated both the demand and the prescription of ARV as PEP and especially the diversity and inconsistency in the criteria used. In this context, in April of 2000 the Centre for Epidemiological Studies on AIDS of Catalonia (CEESCAT) (Department of Health and Social Security of the Autonomous Government of Catalonia), in collaboration with the National AIDS Plan and the AIDS Study Group (GESIDA), promoted the creation of a working group for the drafting of recommendations for PEP against HIV outside the occupational health context. The recommendations have been made bearing in mind the exceptional character of the exposure, the time elapsed since exposure, as well as evaluation of the risk of infection according to the type of exposure and the information available on the source of infection. In addition, the recommendations include the immediate measures necessary, as well as the preventive measures and clinical follow-up required both for HIV and for other infectious agents. All PEP regimens should be started within 72 hours of exposure and appropriate daily doses of two nucleoside reverse transcriptase inhibitors (NRTIs) and a protease inhibitor (PI), or two NRTIs and a non-nucleoside reverse transcriptase inhibitor (NNRTIs), should be administered for four weeks, bearing in mind the pharmacological and clinical situation of the source person. These recommendations should be updated periodically.

New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

PubMed

Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

2012-12-01

HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

Antiretroviral medication prescribing errors are common with hospitalization of HIV-infected patients.

PubMed

Commers, Tessa; Swindells, Susan; Sayles, Harlan; Gross, Alan E; Devetten, Marcel; Sandkovsky, Uriel

2014-01-01

Errors in prescribing antiretroviral therapy (ART) often occur with the hospitalization of HIV-infected patients. The rapid identification and prevention of errors may reduce patient harm and healthcare-associated costs. A retrospective review of hospitalized HIV-infected patients was carried out between 1 January 2009 and 31 December 2011. Errors were documented as omission, underdose, overdose, duplicate therapy, incorrect scheduling and/or incorrect therapy. The time to error correction was recorded. Relative risks (RRs) were computed to evaluate patient characteristics and error rates. A total of 289 medication errors were identified in 146/416 admissions (35%). The most common was drug omission (69%). At an error rate of 31%, nucleoside reverse transcriptase inhibitors were associated with an increased risk of error when compared with protease inhibitors (RR 1.32; 95% CI 1.04-1.69) and co-formulated drugs (RR 1.59; 95% CI 1.19-2.09). Of the errors, 31% were corrected within the first 24 h, but over half (55%) were never remedied. Admissions with an omission error were 7.4 times more likely to have all errors corrected within 24 h than were admissions without an omission. Drug interactions with ART were detected on 51 occasions. For the study population (n = 177), an increased risk of admission error was observed for black (43%) compared with white (28%) individuals (RR 1.53; 95% CI 1.16-2.03) but no significant differences were observed between white patients and other minorities or between men and women. Errors in inpatient ART were common, and the majority were never detected. The most common errors involved omission of medication, and nucleoside reverse transcriptase inhibitors had the highest rate of prescribing error. Interventions to prevent and correct errors are urgently needed.

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease.

PubMed

Jochmans, Dirk; Anders, Maria; Keuleers, Inge; Smeulders, Liesbeth; Kräusslich, Hans-Georg; Kraus, Günter; Müller, Barbara

2010-10-15

Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action.

Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

PubMed Central

2010-01-01

Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1) reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR) related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs) can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM). Specific cytotoxicity was reverted by addition of PR inhibitors. Two of the most active compounds, VRX-480773 and GW-678248, were also tested in primary human cells and mediated cytotoxicity on HIV-1 infected peripheral blood mononuclear cells. Conclusion These data present proof of concept for targeted drug induced elimination of HIV producing cells. While NNRTIs themselves may not be sufficiently potent for therapeutic application, the results provide a basis for the development of drugs exploiting this mechanism of action. PMID:20950436

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

Risk factors and outcomes for the Q151M and T69 insertion HIV-1 resistance mutations in historic UK data.

PubMed

Stirrup, Oliver T; Dunn, David T; Tostevin, Anna; Sabin, Caroline A; Pozniak, Anton; Asboe, David; Cox, Alison; Orkin, Chloe; Martin, Fabiola; Cane, Patricia

2018-04-16

The prevalence of HIV-1 resistance to antiretroviral therapies (ART) has declined in high-income countries over recent years, but drug resistance remains a substantial concern in many low and middle-income countries. The Q151M and T69 insertion (T69i) resistance mutations in the viral reverse transcriptase gene can reduce susceptibility to all nucleoside/tide analogue reverse transcriptase inhibitors, motivating the present study to investigate the risk factors and outcomes associated with these mutations. We considered all data in the UK HIV Drug Resistance Database for blood samples obtained in the period 1997-2014. Where available, treatment history and patient outcomes were obtained through linkage to the UK Collaborative HIV Cohort study. A matched case-control approach was used to assess risk factors associated with the appearance of each of the mutations in ART-experienced patients, and survival analysis was used to investigate factors associated with viral suppression. A further analysis using matched controls was performed to investigate the impact of each mutation on survival. A total of 180 patients with Q151M mutation and 85 with T69i mutation were identified, almost entirely from before 2006. Occurrence of both the Q151M and T69i mutations was strongly associated with cumulative period of virological failure while on ART, and for Q151M there was a particular positive association with use of stavudine and negative association with use of boosted-protease inhibitors. Subsequent viral suppression was negatively associated with viral load at sequencing for both mutations, and for Q151M we found a negative association with didanosine use but a positive association with boosted-protease inhibitor use. The results obtained in these analyses were also consistent with potentially large associations with other drugs. Analyses were inconclusive regarding associations between the mutations and mortality, but mortality was high for patients with low CD4 at detection. The Q151M and T69i resistance mutations are now very rare in the UK. Our results suggest that good outcomes are possible for people with these mutations. However, in this historic sample, viral load and CD4 at detection were important factors in determining prognosis.

Phylogenetic analysis of HIV-1 reverse transcriptase sequences from 382 patients recruited in JJ Hospital of Mumbai, India, between 2002 and 2008.

PubMed

Deshpande, Alaka; Jauvin, Valerie; Pinson, Patricia; Jeannot, Anne Cecile; Fleury, Herve J

2009-06-01

Analysis of reverse transcriptase (RT) sequences of 382 HIV-1 isolates from untreated and treated patients recruited in JJ Hospital (Mumbai, India) between 2002 and 2008 shows that subtype C is largely predominant (98%) and that non-C sequences cluster with A1, B, CRF01_AE, and CRF06_cpx.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice.

PubMed

Huang, Chun; Li, Runqin; Zhang, Yinglin; Gong, Jianping

2017-10-01

Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells.

Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

PubMed Central

Li, Runqin; Zhang, Yinglin

2016-01-01

Background and Objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. Materials and Methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells. PMID:27402632

Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

PubMed Central

2011-01-01

Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

Effects of Hypomagnetic Conditions and Reversed Geomagnetic Field on Calcium-Dependent Proteases of Invertebrates and Fish

NASA Astrophysics Data System (ADS)

Kantserova, N. P.; Krylov, V. V.; Lysenko, L. A.; Ushakova, N. V.; Nemova, N. N.

2017-12-01

The effects of hypomagnetic conditions and the reversal of the geomagnetic field (GMF) on intracellular Ca2+-dependent proteases (calpains) of fish and invertebrates have been studied in vivo and in vitro. It is found that the intravital exposure of examined animals to hypomagnetic conditions leads to a significant decrease in its calpain activity. The activity of preparations of calcium-dependent proteases was tested in separate experiments. It is shown that preparations of Ca2+-dependent proteases from invertebrates and fish are also inactivated substantially under effect of hypomagnetic conditions. The ambiguous results obtained in the experiments with a reversed GMF do not make it possible to discuss the biological response of calcium-dependent proteases to the reversal of the GMF.

Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-03-01

Over the past 10 years, several anti-viral drugs have become available to fight the HIV infection. Antiretroviral treatment reduces the mortality of AIDS. Nonnucleoside inhibitors of HIV-1 reverse transcriptase are specific and potentially nontoxic drugs against AIDS. The crystal structures of five nonnucleoside inhibitors of HIV-1 reverse transcriptase are presented here. The structural parameters, especially those describing the angular orientation of the π-electron systems and influencing biological activity, were determined for all of the investigated inhibitors. The chemical character and orientation of the substituent at C4 position of the benzimidazole moiety substantially influences the anti-viral activity. The structural data of the investigated inhibitors is a good basis for modeling enzyme-inhibitor interactions for structure-assisted drug design.

Virtual screening studies on HIV-1 reverse transcriptase inhibitors to design potent leads.

PubMed

Vadivelan, S; Deeksha, T N; Arun, S; Machiraju, Pavan Kumar; Gundla, Rambabu; Sinha, Barij Nayan; Jagarlapudi, Sarma A R P

2011-03-01

The purpose of this study is to identify novel and potent inhibitors against HIV-1 reverse transcriptase (RT). The crystal structure of the most active ligand was converted into a feature-shaped query. This query was used to align molecules to generate statistically valid 3D-QSAR (r(2) = 0.873) and Pharmacophore models (HypoGen). The best HypoGen model consists of three Pharmacophore features (one hydrogen bond acceptor, one hydrophobic aliphatic and one ring aromatic) and further validated using known RT inhibitors. The designed novel inhibitors are further subjected to docking studies to reduce the number of false positives. We have identified and proposed some novel and potential lead molecules as reverse transcriptase inhibitors using analog and structure based studies. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

PubMed Central

Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

2011-01-01

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

Combination nucleoside/nucleotide reverse transcriptase inhibitors for treatment of HIV infection.

PubMed

Akanbi, Maxwell O; Scarsi, Kimberly K; Scarci, Kimberly; Taiwo, Babafemi; Murphy, Robert L

2012-01-01

The combination of two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs) and a third agent from another antiretroviral class is currently recommended for initial antiretroviral therapy. In general, N(t)RTIs remain relevant in subsequent regimens. There are currently six nucleoside reverse transcriptase inhibitors and one nucleotide reverse transcriptase inhibitor drug entities available, and several formulations that include two or more N(t)RTIs in a fixed-dose combination. These entities have heterogeneous pharmacological and clinical properties. Accordingly, toxicity, pill burden, dosing frequency, potential drug-drug interaction, preexisting antiretroviral drug resistance and comorbid conditions should be considered when constructing a regimen. This approach is critical in order to optimize virologic efficacy and clinical outcomes. This article reviews N(t)RTI combinations used in the treatment of HIV-infected adults. The pharmacological properties of each N(t)RTI, and the clinical trials that have influenced treatment guidelines are discussed. It is likely that N(t)RTIs will continue to dominate the global landscape of HIV treatment and prevention, despite emerging interest in N(t)RTI-free combination therapy. Clinical domains where only few alternatives to N(t)RTIs exist include treatment of HIV/HBV coinfection and HIV-2. There is a need for novel N(t)RTIs with enhanced safety and resistance profiles compared with current N(t)RTIs.

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

The Role of eIF4E Activity in Breast Cancer

DTIC Science & Technology

2010-08-01

ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less product...have previously shown that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

Assessment of second-line antiretroviral regimens for HIV therapy in Africa.

PubMed

Paton, Nicholas I; Kityo, Cissy; Hoppe, Anne; Reid, Andrew; Kambugu, Andrew; Lugemwa, Abbas; van Oosterhout, Joep J; Kiconco, Mary; Siika, Abraham; Mwebaze, Raymond; Abwola, Mary; Abongomera, George; Mweemba, Aggrey; Alima, Hillary; Atwongyeire, Dickens; Nyirenda, Rose; Boles, Justine; Thompson, Jennifer; Tumukunde, Dinah; Chidziva, Ennie; Mambule, Ivan; Arribas, Jose R; Easterbrook, Philippa J; Hakim, James; Walker, A Sarah; Mugyenyi, Peter

2014-07-17

The efficacy and toxic effects of nucleoside reverse-transcriptase inhibitors (NRTIs) are uncertain when these agents are used with a protease inhibitor in second-line therapy for human immunodeficiency virus (HIV) infection in resource-limited settings. Removing the NRTIs or replacing them with raltegravir may provide a benefit. In this open-label trial in sub-Saharan Africa, we randomly assigned 1277 adults and adolescents with HIV infection and first-line treatment failure to receive a ritonavir-boosted protease inhibitor (lopinavir-ritonavir) plus clinician-selected NRTIs (NRTI group, 426 patients), a protease inhibitor plus raltegravir in a superiority comparison (raltegravir group, 433 patients), or protease-inhibitor monotherapy after 12 weeks of induction therapy with raltegravir in a noninferiority comparison (monotherapy group, 418 patients). The primary composite end point, good HIV disease control, was defined as survival with no new World Health Organization stage 4 events, a CD4+ count of more than 250 cells per cubic millimeter, and a viral load of less than 10,000 copies per milliliter or 10,000 copies or more with no protease resistance mutations at week 96 and was analyzed with the use of imputation of data (≤4%). Good HIV disease control was achieved in 60% of the patients (mean, 255 patients) in the NRTI group, 64% of the patients (mean, 277) in the raltegravir group (P=0.21 for the comparison with the NRTI group; superiority of raltegravir not shown), and 55% of the patients (mean, 232) in the monotherapy group (noninferiority of monotherapy not shown, based on a 10-percentage-point margin). There was no significant difference in rates of grade 3 or 4 adverse events among the three groups (P=0.82). The viral load was less than 400 copies per milliliter in 86% of patients in the NRTI group, 86% in the raltegravir group (P=0.97), and 61% in the monotherapy group (P<0.001). When given with a protease inhibitor in second-line therapy, NRTIs retained substantial virologic activity without evidence of increased toxicity, and there was no advantage to replacing them with raltegravir. Virologic control was inferior with protease-inhibitor monotherapy. (Funded by European and Developing Countries Clinical Trials Partnership and others; EARNEST Current Controlled Trials number, ISRCTN37737787, and ClinicalTrials.gov number, NCT00988039.).

High rates of virological failure and drug resistance in perinatally HIV-1-infected children and adolescents receiving lifelong antiretroviral therapy in routine clinics in Togo

PubMed Central

Salou, Mounerou; Dagnra, Anoumou Y; Butel, Christelle; Vidal, Nicole; Serrano, Laetitia; Takassi, Elom; Konou, Abla A; Houndenou, Spero; Dapam, Nina; Singo-Tokofaï, Assetina; Pitche, Palokinam; Atakouma, Yao; Prince-David, Mireille; Delaporte, Eric; Peeters, Martine

2016-01-01

Introduction Antiretroviral treatment (ART) has been scaled up over the last decade but compared to adults, children living with HIV are less likely to receive ART. Moreover, children and adolescents are more vulnerable than adults to virological failure (VF) and emergence of drug resistance. In this study we determined virological outcome in perinatally HIV-1-infected children and adolescents receiving ART in Togo. Methods HIV viral load (VL) testing was consecutively proposed to all children and adolescents who were on ART for at least 12 months when attending HIV healthcare services for their routine follow-up visit (June to September 2014). Plasma HIV-1 VL was measured using the m2000 RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA). Genotypic drug resistance was done for all samples with VL>1000 copies/ml. Results and discussion Among 283 perinatally HIV-1-infected children and adolescents included, 167 (59%) were adolescents and 116 (41%) were children. The median duration on ART was 48 months (interquartile range: 28 to 68 months). For 228 (80.6%), the current ART combination consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) (zidovudine and lamivudine) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine or efavirenz). Only 28 (9.9%) were on a protease inhibitor (PI)-based regimen. VL was below the detection limit (i.e. 40 copies/ml) for 102 (36%), between 40 and 1000 copies/ml for 35 (12.4%) and above 1000 copies/ml for 146 (51.6%). Genotypic drug-resistance testing was successful for 125/146 (85.6%); 110/125 (88.0%) were resistant to both NRTIs and NNRTIs, 1/125 (0.8%) to NRTIs only, 4/125 (3.2%) to NNRTIs only and three harboured viruses resistant to reverse transcriptase and PIs. Overall, 86% (108/125) of children and adolescents experiencing VF and successfully genotyped, corresponding thus to at least 38% of the study population, had either no effective ART or had only a single effective drug in their current ART regimen. Conclusions Our study provided important information on virological outcome on lifelong ART in perinatally HIV-1-infected children and adolescents who were still on ART and continued to attend antiretroviral (ARV) clinics for follow-up visits. Actual conditions for scaling up and monitoring lifelong ART in children in resource-limited countries can have dramatic long-term outcomes and illustrate that paediatric ART receives inadequate attention. PMID:27125320

The public health approach to identify antiretroviral therapy failure: high-level nucleoside reverse transcriptase inhibitor resistance among Malawians failing first-line antiretroviral therapy

PubMed Central

Hosseinipour, Mina C.; van Oosterhout, Joep J.G.; Weigel, Ralf; Phiri, Sam; Kamwendo, Debbie; Parkin, Neil; Fiscus, Susan A.; Nelson, Julie A.E.; Eron, Joseph J.; Kumwenda, Johnstone

2010-01-01

Background Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi. Methods Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed. Results Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine. Conclusion When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART. PMID:19417582

The Role of elF4E Activity in Breast Cancer

DTIC Science & Technology

2011-08-01

protein; ORF, open reading frame; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase ; uORF, upstream ORF; UTR...Reactions were also performed using template lacking RT ( reverse transcriptase ): products were either undetectable or greatly reduced (>30000-fold less...that a 5’UTR expressed from the human AXIN2 gene contains a sixty nucleotide sequence that is predicted to form a stable stem-loop structure6. This

Molecular docking and 3D-QSAR studies on triazolinone and pyridazinone, non-nucleoside inhibitor of HIV-1 reverse transcriptase.

PubMed

Sivan, Sree Kanth; Manga, Vijjulatha

2010-06-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV-1 reverse transcriptase. Recently a series of Triazolinone and Pyridazinone were reported as potent inhibitors of HIV-1 wild type reverse transcriptase. In the present study, docking and 3D quantitative structure activity relationship (3D QSAR) studies involving comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 31 molecules. Ligands were built and minimized using Tripos force field and applying Gasteiger-Hückel charges. These ligands were docked into protein active site using GLIDE 4.0. The docked poses were analyzed; the best docked poses were selected and aligned. CoMFA and CoMSIA fields were calculated using SYBYL6.9. The molecules were divided into training set and test set, a PLS analysis was performed and QSAR models were generated. The model showed good statistical reliability which is evident from the r2 nv, q2 loo and r2 pred values. The CoMFA model provides the most significant correlation of steric and electrostatic fields with biological activities. The CoMSIA model provides a correlation of steric, electrostatic, acceptor and hydrophobic fields with biological activities. The information rendered by 3D QSAR model initiated us to optimize the lead and design new potential inhibitors.

The Reverse Transcriptase of the Tf1 Retrotransposon Has a Specific Novel Activity for Generating the RNA Self-Primer That Is Functional in cDNA Synthesis▿

PubMed Central

Hizi, Amnon

2008-01-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5′ end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3′ end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs. PMID:18753200

The reverse transcriptase of the Tf1 retrotransposon has a specific novel activity for generating the RNA self-primer that is functional in cDNA synthesis.

PubMed

Hizi, Amnon

2008-11-01

The Tf1 retrotransposon of Schizosaccharomyces pombe represents a group of eukaryotic long terminal repeat (LTR) retroelements that, based on their sequences, were predicted to use an RNA self-primer for initiating reverse transcription while synthesizing the negative-sense DNA strand. This feature is substantially different from the one typical to retroviruses and other LTR retrotransposons that all exhibit a tRNA-dependent priming mechanism. Genetic studies have suggested that the self-primer of Tf1 can be generated by a cleavage between the 11th and 12th bases of the Tf1 RNA transcript. The in vitro data presented here show that recombinant Tf1 reverse transcriptase indeed introduces a nick at the end of a duplexed region at the 5' end of Tf1 genomic RNA, substantiating the prediction that this enzyme is responsible for generating this RNA self-primer. The 3' end of the primer, generated in this manner, can then be extended upon the addition of deoxynucleoside triphosphates by the DNA polymerase activity of the same enzyme, synthesizing the negative-sense DNA strand. This functional primer must have been generated by the RNase H activity of Tf1 reverse transcriptase, since a mutant enzyme lacking this activity has lost its ability to generate the self-primer. It was also found here that the reverse transcriptases of human immunodeficiency virus type 1 and of murine leukemia virus do not exhibit this specific cleavage activity. In all, it is likely that the observed unique mechanism of self-priming in Tf1 represents an early advantageous form of initiating reverse transcription in LTR retroelements without involving cellular tRNAs.

Comparison of HIV-1 pol and env sequences of blood, CSF, brain and spleen isolates collected ante-mortem and post-mortem.

PubMed

Caragounis, E-C; Gisslén, M; Lindh, M; Nordborg, C; Westergren, S; Hagberg, L; Svennerholm, B

2008-02-01

HIV-1 infects the central nervous system (CNS) early in the course of infection. However, it is not known to what extent the virus evolves independently within the CNS and whether the HIV-RNA in cerebrospinal fluid (CSF) reflects the viral population replicating within the brain parenchyma or the systemic infection. The aim of this study was to investigate HIV-1 evolution in the CNS and the origin of HIV-1 in CSF. Longitudinally derived paired blood and CSF samples and post-mortem samples from CSF, brain and spleen were collected over a period of up to 63 months from three HIV-1 infected men receiving antiretroviral treatment and presenting with symptoms of AIDS dementia complex (ADC). Phylogenetic analyses of HIV-1 V3, reverse transcriptase (RT) and protease sequences from patient isolates suggest compartmentalization with distinct viral strains in blood, CSF and brain. We found a different pattern of RT and accessory protease mutations in the systemic infection compared to the CNS. We conclude that HIV-1 may to some extent evolve independently in the CNS and the viral population in CSF mainly reflects the infection in the brain parenchyma in patients with ADC. This is of importance in understanding HIV pathogenesis and can have implications on treatment of HIV-1 patients.

Molecular epidemiological analysis of env and pol sequences in newly diagnosed HIV type 1-infected, untreated patients in Hungary.

PubMed

Mezei, Mária; Ay, Eva; Koroknai, Anita; Tóth, Renáta; Balázs, Andrea; Bakos, Agnes; Gyori, Zoltán; Bánáti, Ferenc; Marschalkó, Márta; Kárpáti, Sarolta; Minárovits, János

2011-11-01

The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008-2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classification of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

Broadening the use of antiretroviral therapy: the case for feline leukemia virus

PubMed Central

Greggs, Willie M; Clouser, Christine L; Patterson, Steven E; Mansky, Louis M

2011-01-01

Antiretroviral drugs have saved and extended the lives of millions of individuals infected with HIV. The major classes of anti-HIV drugs include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and entry/fusion inhibitors. While antiretroviral drug regimens are not commonly used to treat other types of retroviral infections, there are instances where there is a perceived need for re-evaluation of the benefits of antiretroviral therapy. One case in point is that of feline leukemia virus (FeLV), an infection of companion felines. While vaccines exist to prevent FeLV infection and spread, they have not eliminated FeLV infection. For FeLV-infected felines and their human companions, antiretroviral therapy would be desirable and of practical importance if good options were available. Here, we discuss FeLV biology and current treatment options, and propose that there is a need for antiretroviral treatment options for FeLV infection. The comparative use and analysis of antiretroviral therapy can provide new insights into the mechanism of antiretroviral drug action. PMID:21479142

HIV-associated lipodystrophy syndrome: A review of clinical aspects

PubMed Central

Baril, Jean-Guy; Junod, Patrice; LeBlanc, Roger; Dion, Harold; Therrien, Rachel; Laplante, François; Falutz, Julian; Côté, Pierre; Hébert, Marie-Nicole; Lalonde, Richard; Lapointe, Normand; Lévesque, Dominic; Pinault, Lyse; Rouleau, Danielle; Tremblay, Cécile; Trottier, Benoît; Trottier, Sylvie; Tsoukas, Chris; Weiss, Karl

2005-01-01

Approximately two years after the introduction of highly active antiretroviral therapy for the treatment of HIV infection, body shape changes and metabolic abnormalities were increasingly observed. Initially, these were ascribed to protease inhibitors, but it is now clear that nucleoside reverse transcriptase inhibitors also contribute to lipodystrophy syndrome. The syndrome groups together clinical conditions describing changes in body fat distribution that include lipoatrophy, lipoaccumulation or both. However, there does not appear to be a direct link between lipoatrophy and lipoaccumulation that would support a single mechanism for the redistribution of body fat. Currently, there is no clear definition of lipodystrophy, which explains the difficulty in determining its prevalence and etiology. There are no current guidelines for the treatment of fat distribution abnormalities that occur in the absence of other metabolic complications. The present article reviews the current state of knowledge of the definition, symptoms, risk factors, pathogenesis, diagnosis and treatment of the morphological changes associated with lipodystrophy syndrome. PMID:18159551

Fosamprenavir calcium plus ritonavir for HIV infection.

PubMed

Torres, Harrys A; Arduino, Roberto C

2007-06-01

Fosamprenavir is a protease inhibitor (PI) approved for the treatment of HIV-1 infection. Fosamprenavir is a prodrug of amprenavir developed to reduce the pill burden yet maintain the unique resistance pattern and efficacy associated with amprenavir. In a head-to-head, noninferiority trial in antiretroviral treatment-naive HIV-infected patients, the antiviral efficacy and tolerability of ritonavir-boosted fosamprenavir was not inferior to ritonavir-boosted lopinavir, when the PIs were combined with two other nucleoside reverse transcriptase inhibitors. There are fewer studies published about fosamprenavir use in antiretroviral treatment-experienced HIV-infected patients. The high genetic barrier to the development of resistance to fosamprenavir and the low level of cross-resistance between ritonavir-boosted fosamprenavir and other PI regimens are notable. As with amprenavir, gastrointestinal disturbance and rash are the most frequent short-term treatment-limiting events with fosamprenavir. Treatment with ritonavir-boosted fosamprenavir can produce a durable response. To date, fosamprenavir is one of the recommended preferred PI components for the treatment of antiretroviral-naive HIV-infected patients.

Genetic Diversity of HIV-1 in Tunisia.

PubMed

El Moussi, Awatef; Thomson, Michael M; Delgado, Elena; Cuevas, María Teresa; Nasr, Majda; Abid, Salma; Ben Hadj Kacem, Mohamed Ali; Benaissa Tiouiri, Hanene; Letaief, Amel; Chakroun, Mohamed; Ben Jemaa, Mounir; Hamdouni, Hayet; Tej Dellagi, Rafla; Kheireddine, Khaled; Boutiba, Ilhem; Pérez-Álvarez, Lucía; Slim, Amine

2017-01-01

In this study, the genetic diversity of HIV-1 in Tunisia was analyzed. For this, 193 samples were collected in different regions of Tunisia between 2012 and 2015. A protease and reverse transcriptase fragment were amplified and sequenced. Phylogenetic analyses were performed through maximum likelihood and recombination was analyzed by bootscanning. Six HIV-1 subtypes (B, A1, G, D, C, and F2), 5 circulating recombinant forms (CRF02_AG, CRF25_cpx, CRF43_02G, CRF06_cpx, and CRF19_cpx), and 11 unique recombinant forms were identified. Subtype B (46.4%) and CRF02_AG (39.4%) were the predominant genetic forms. A group of 44 CRF02_AG sequences formed a distinct Tunisian cluster, which also included four viruses from western Europe. Nine viruses were closely related to isolates collected in other African or in European countries. In conclusion, a high HIV-1 genetic diversity is observed in Tunisia and the local spread of CRF02_AG is first documented in this country.

The impact of HIV-1 reverse transcriptase polymorphisms on responses to first-line nonnucleoside reverse transcriptase inhibitor-based therapy in HIV-1-infected adults.

PubMed

Mackie, Nicola E; Dunn, David T; Dolling, David; Garvey, Lucy; Harrison, Linda; Fearnhill, Esther; Tilston, Peter; Sabin, Caroline; Geretti, Anna M

2013-09-10

HIV-1 genetic variability may influence antiretroviral therapy (ART) outcomes. The study aim was to determine the impact of polymorphisms in regions known to harbor major nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations (codons 90-108, 135-138, 179-190, 225-348) on virologic responses to first-line NNRTI-based ART. Reverse transcriptase sequences from ART-naive individuals who commenced efavirenz (EFV) or nevirapine (NVP) with at least two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) without major drug resistance mutations were analyzed. The impact of polymorphisms on week 4 viral load decrease and time to virologic failure was measured over a median 97 weeks. Among 4528 patients, most were infected with HIV-1 subtype B (67%) and commenced EFV-based ART (84%). Overall, 2598 (57%) had at least one polymorphism, most frequently at codons 90, 98, 101, 103, 106, 135, 138, 179, and 238. Virologic failure rates were increased in patients with two (n = 597) or more than two (n = 72) polymorphisms [adjusted hazard ratio 1.43; 95% confidence interval (CI) 1.07-1.92; P = 0.016]. Polymorphisms associated with virologic failure occurred at codons 90 (mostly V90I), 98 (mostly A98S), and 103 (mostly K103R), with adjusted hazard ratios of 1.78 (1.15-2.73; P = 0.009), 1.55 (1.16-2.08; P = 0.003), and 1.75 (1.00-3.05: P = 0.049), respectively. Polymorphisms at codon 179, especially V179D/E/T, predicted reduced week 4 responses (P = 0.001) but not virologic failure. The occurrence of multiple polymorphisms, though uncommon, was associated with a small increase in the risk of NNRTI treatment failure; significant effects were seen with polymorphisms at codon 90, 98, and 103. The mechanisms underlying the slower suppression seen with V179D/E/T deserve further investigation.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

High Prevalence of HIV Drug Resistance Among Newly Diagnosed Infants Aged <18 Months: Results From a Nationwide Surveillance in Nigeria.

PubMed

Inzaule, Seth C; Osi, Samuels J; Akinbiyi, Gbenga; Emeka, Asadu; Khamofu, Hadiza; Mpazanje, Rex; Ilesanmi, Oluwafunke; Ndembi, Nicaise; Odafe, Solomon; Sigaloff, Kim C E; Rinke de Wit, Tobias F; Akanmu, Sulaimon

2018-01-01

WHO recommends protease-inhibitor-based first-line regimen in infants because of risk of drug resistance from failed prophylaxis used in prevention of mother-to-child transmission (PMTCT). However, cost and logistics impede implementation in sub-Saharan Africa, and >75% of children still receive nonnucleoside reverse transcriptase inhibitor-based regimen (NNRTI) used in PMTCT. We assessed the national pretreatment drug resistance prevalence of HIV-infected children aged <18 months in Nigeria, using WHO-recommended HIV drug resistance surveillance protocol. We used remnant dried blood spots collected between June 2014 and July 2015 from 15 early infant diagnosis facilities spread across all the 6 geopolitical regions of Nigeria. Sampling was through a probability proportional-to-size approach. HIV drug resistance was determined by population-based sequencing. Overall, in 48% of infants (205 of 430) drug resistance mutations (DRM) were detected, conferring resistance to predominantly NNRTIs (45%). NRTI and multiclass NRTI/NNRTI resistance were present at 22% and 20%, respectively, while resistance to protease inhibitors was at 2%. Among 204 infants with exposure to drugs for PMTCT, 57% had DRMs, conferring NNRTI resistance in 54% and multiclass NRTI/NNRTI resistance in 29%. DRMs were also detected in 34% of 132 PMTCT unexposed infants. A high frequency of PDR, mainly NNRTI-associated, was observed in a nationwide surveillance among newly diagnosed HIV-infected children in Nigeria. PDR prevalence was equally high in PMTCT-unexposed infants. Our results support the use of protease inhibitor-based first-line regimens in HIV-infected young children regardless of PMTCT history and underscore the need to accelerate implementation of the newly disseminated guideline in Nigeria.

Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing.

PubMed

Tzou, Philip L; Ariyaratne, Pramila; Varghese, Vici; Lee, Charlie; Rakhmanaliev, Elian; Villy, Carolin; Yee, Meiqi; Tan, Kevin; Michel, Gerd; Pinsky, Benjamin A; Shafer, Robert W

2018-06-01

The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy. Copyright © 2018 American Society for Microbiology.

Prevalence and predictors of antiretroviral drug resistance in newly diagnosed HIV-1 infection.

PubMed

Booth, Clare L; Garcia-Diaz, Ana M; Youle, Michael S; Johnson, Margaret A; Phillips, Andrew; Geretti, Anna Maria

2007-03-01

To determine prevalence and predictors of antiretroviral drug resistance in newly diagnosed individuals with HIV-1 infection, using a systematic approach to avoid selection bias. Plasma samples from all persons diagnosed HIV-1 seropositive at a large London centre between April 2004 and February 2006 underwent sequencing of HIV-1 reverse transcriptase (RT) and protease genes. Subtype was assigned by phylogenetic analysis. Resistance was scored according to the IAS-USA list (2005) modified to include T215revertants and exclude isolated E44D or V118I and minor protease mutations. Recent seroconversion was identified by HIV antibody avidity testing. The cohort of 239 included 169 (70.7%) males, 126 (52.7%) homosexuals, 118 (49.5%) persons of white ethnicity and 144 (60.0%) persons born outside the UK. Subtypes included B 134 (56.1%), C 46 (19.2%), A 17 (7.1%), other non-B 42 (17.6%). The prevalence of resistance mutations was 17/239 (7.1%; 95% confidence interval 4.5-11.1%), comprising 10/239 (4.2%) nucleoside/nucleotide RT inhibitor (NRTI); 4/239 (1.7%) non-nucleoside RT inhibitor (NNRTI) and 4/239 (1.7%) protease inhibitor (PI) associated mutations. Dual-class (NRTI + PI) resistance mutations were detected in 1/239 (0.4%) person. The prevalence of resistance mutations was 7/85 (8.2%) and 10/154 (6.5%) in persons with recent and established infection, respectively. In multivariate analysis, having been born in the UK and high CD4 count, but not gender, age, risk group, ethnicity or subtype, were independent predictors of resistance. In an unselected UK cohort, subtypes other than B accounted for 43.9% of new HIV-1 diagnoses. The prevalence of resistance mutations was 7.1% and highest in those born in the UK.

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

Evaluation and Management of Dyslipidemia in Patients with HIV Infection

PubMed Central

Green, Michael L

2002-01-01

OBJECTIVE Persons with HIV infection develop metabolic abnormalities related to their antiretroviral therapy and HIV infection itself. The objective of this study was to summarize the emerging evidence for the incidence, etiology, health risks, and treatment of dyslipidemias in HIV disease. DESIGN Systematic review of original research with quantitative synthesis. MAIN RESULTS Dyslipidemia is common in persons with HIV infection on highly active antiretroviral therapy (HAART), but methodologic differences between studies preclude precise estimates of prevalence and incidence. The typical pattern includes elevated total cholesterol, low-density lipoprotein cholesterol, and triglycerides, which may be markedly elevated. The dyslipidemia may be associated with lipodystrophy, insulin resistance, and, rarely, frank diabetes mellitus. Exposure to protease inhibitors (PIs) is associated with this entire range of metabolic abnormalities. PI-naïve patients on nucleoside reverse transcriptase inhibitors (NRTIs) may develop lipodystrophy, insulin resistance, hypercholesterolemia, and possibly modest elevations in triglycerides but not severe hypertriglyceridemia, which appears to be linked to PIs alone. Most studies have not found an association between CD4 lymphocyte count or HIV viral load and lipid abnormalities. The pathogenesis is incompletely understood and appears to be multifactorial. There are insufficient data to definitively support an increased coronary heart disease risk in patients with HIV-related dyslipidemia. However, some of the same metabolic abnormalities remain firmly established risk factors in other populations. Patients on HAART with severe hypertriglyceridemia may develop pancreatitis or other manifestations of the chylomicronemia syndrome. Some of the metabolic derangements (particularly hypertriglyceridemia) may improve upon replacing a PI with a non-nucleoside reverse transcriptase inhibitor. The limited experience suggests that fibrates, pravastatin, and atorvastatin can safely treat lipid abnormalities in HIV-infected patients. CONCLUSIONS Patients with HIV infection on HAART should be screened for lipid disorders, given their incidence, potential for morbidity, and possible long-term cardiovascular risk. Treatment decisions are complex and must include assessments of cardiac risk, HIV infection status, reversibility of the dyslipidemia, and the effectiveness and toxicities of lipid-lowering medications. The multiple potential drug interactions with antiretroviral or other HIV-related medications should be considered in lipid-lowering drug selection and monitoring. PMID:12390557

Murine Leukemia Virus Reverse Transcriptase: Structural Comparison with HIV-1 Reverse Transcriptase

PubMed Central

Coté, Marie L.; Roth, Monica J.

2008-01-01

Recent X-ray crystal structure determinations of Moloney murine leukemia virus reverse transcriptase (MoMLV RT) have allowed for more accurate structure/function comparisons to HIV-1 RT than were formerly possible. Previous biochemical studies of MoMLV RT in conjunction with knowledge of sequence homologies to HIV-1 RT and overall fold similarities to RTs in general, provided a foundation upon which to build. In addition, numerous crystal structures of the MoMLV RT fingers/palm subdomain had also shed light on one of the critical functions of the enzyme, specifically polymerization. Now in the advent of new structural information, more intricate examination of MoMLV RT in its entirety can be realized, and thus the comparisons with HIV-1 RT may be more critically elucidated. Here, we will review the similarities and differences between MoMLV RT and HIV-1 RT via structural analysis, and propose working models for the MoMLV RT based upon that information. PMID:18294720

Long-term effectiveness of initiating non-nucleoside reverse transcriptase inhibitor- versus ritonavir-boosted protease inhibitor-based antiretroviral therapy: implications for first-line therapy choice in resource-limited settings

PubMed Central

Lima, Viviane D; Hull, Mark; McVea, David; Chau, William; Harrigan, P Richard; Montaner, Julio SG

2016-01-01

Introduction In many resource-limited settings, combination antiretroviral therapy (cART) failure is diagnosed clinically or immunologically. As such, there is a high likelihood that patients may stay on a virologically failing regimen for a substantial period of time. Here, we compared the long-term impact of initiating non-nucleoside reverse transcriptase inhibitor (NNRTI)- versus boosted protease inhibitor (bPI)-based cART in British Columbia (BC), Canada. Methods We followed prospectively 3925 ART-naïve patients who started NNRTIs (N=1963, 50%) or bPIs (N=1962; 50%) from 1 January 2000 until 30 June 2013 in BC. At six months, we assessed whether patients virologically failed therapy (a plasma viral load (pVL) >50 copies/mL), and we stratified them based on the pVL at the time of failure ≤500 versus >500 copies/mL. We then followed these patients for another six months and calculated their probability of achieving subsequent viral suppression (pVL <50 copies/mL twice consecutively) and of developing drug resistance. These probabilities were adjusted for fixed and time-varying factors, including cART adherence. Results At six months, virologic failure rates were 9.5 and 14.3 cases per 100 person-months for NNRTI and bPI initiators, respectively. NNRTI initiators who failed with a pVL ≤500 copies/mL had a 16% higher probability of achieving subsequent suppression at 12 months than bPI initiators (0.81 (25th–75th percentile 0.75–0.83) vs. 0.72 (0.61–0.75)). However, if failing NNRTI initiators had a pVL >500 copies/mL, they had a 20% lower probability of suppressing at 12 months than pVL-matched bPI initiators (0.37 (0.29–0.45) vs. 0.46 (0.38–0.54)). In terms of evolving HIV drug resistance, those who failed on NNRTI performed worse than bPI in all scenarios, especially if they failed with a viral load >500 copies/mL. Conclusions Our results show that patients who virologically failed at six months on NNRTI and continued on the same regimen had a lower probability of subsequently achieving viral suppression and a higher chance of evolving HIV drug resistance. These results suggest that improving access to regular virologic monitoring is critically important, especially if NNRTI-based cART is to remain a preferred choice for first-line therapy in resource-limited settings. PMID:27499064

Altered Oligodendrocyte Maturation and Myelin Maintenance: The Role of Antiretrovirals in HIV-Associated Neurocognitive Disorders.

PubMed

Jensen, Brigid K; Monnerie, Hubert; Mannell, Maggie V; Gannon, Patrick J; Espinoza, Cagla Akay; Erickson, Michelle A; Bruce-Keller, Annadora J; Gelman, Benjamin B; Briand, Lisa A; Pierce, R Christopher; Jordan-Sciutto, Kelly L; Grinspan, Judith B

2015-11-01

Despite effective viral suppression through combined antiretroviral therapy (cART), approximately half of HIV-positive individuals have HIV-associated neurocognitive disorders (HAND). Studies of antiretroviral-treated patients have revealed persistent white matter abnormalities including diffuse myelin pallor, diminished white matter tracts, and decreased myelin protein mRNAs. Loss of myelin can contribute to neurocognitive dysfunction because the myelin membrane generated by oligodendrocytes is essential for rapid signal transduction and axonal maintenance. We hypothesized that myelin changes in HAND are partly due to effects of antiretroviral drugs on oligodendrocyte survival and/or maturation. We showed that primary mouse oligodendrocyte precursor cell cultures treated with therapeutic concentrations of HIV protease inhibitors ritonavir or lopinavir displayed dose-dependent decreases in oligodendrocyte maturation; however, this effect was rapidly reversed after drug removal. Conversely, nucleoside reverse transcriptase inhibitor zidovudine had no effect. Furthermore, in vivo ritonavir administration to adult mice reduced frontal cortex myelin protein levels. Finally, prefrontal cortex tissue from HIV-positive individuals with HAND on cART showed a significant decrease in myelin basic protein compared with untreated HIV-positive individuals with HAND or HIV-negative controls. These findings demonstrate that antiretrovirals can impact myelin integrity and have implications for myelination in juvenile HIV patients and myelin maintenance in adults on lifelong therapy.

Time trends in drug resistant HIV-1 infections in the United Kingdom up to 2009: multicentre observational study.

PubMed

Dolling, David; Sabin, Caroline; Delpech, Valerie; Smit, Erasmus; Pozniak, Anton; Asboe, David; Brown, Andrew Leigh; Churchill, Duncan; Williams, Ian; Geretti, Anna Maria; Phillips, Andrew; Mackie, Nicola; Murphy, Gary; Castro, Hannah; Pillay, Deenan; Cane, Patricia; Dunn, David; Dolling, David

2012-08-21

To evaluate whether the prevalence of HIV-1 transmitted drug resistance has continued to decline in infections probably acquired within the United Kingdom. Multicentre observational study. All UK public laboratories conducting tests for genotypic HIV resistance as a part of routine care. 14,584 patients infected with HIV-1 subtype B virus, who were first tested for resistance before receiving antiretroviral therapy between January 2002 and December 2009. Prevalence of transmitted drug resistance, defined as one or more resistance mutations from the surveillance list recommended by the World Health Organization. 1654 (11.3%, 95% confidence interval 10.8% to 11.9%) patients had one or more mutations associated with transmitted HIV-1 drug resistance; prevalence was found to decline from 15.5% in 2002 to 9.6% in 2007, followed by a slight increase to 10.9% in 2009 (P=0.21). This later rise was mainly a result of increases in resistance to nucleos(t)ide reverse transcriptase inhibitors (from 5.4% in 2007 to 6.6% in 2009, P=0.24) and protease inhibitors (1.5% to 2.1%, P=0.12). Thymidine analogue mutations, including T215 revertants, remained the most frequent mutations associated with nucleos(t)ide reverse transcriptase inhibitors, despite a considerable fall in stavudine and zidovudine use between 2002 and 2009 (from 29.4% of drug regimens in 2002 to 0.8% in 2009, from 47.9% to 8.8%, respectively). The previously observed decline in the prevalence of transmitted drug resistance in HIV-1 infections probably acquired in the UK seems to have stabilised. The continued high prevalence of thymidine analogue mutations suggests that the source of this resistance may be increasingly from patients who have not undergone antiretroviral therapy and who harbour resistant viruses. Testing of all newly diagnosed HIV-1 positive people should be continued.

2004: which HIV-1 drug resistance mutations are common in clinical practice?

PubMed

Cheung, Peter K; Wynhoven, Brian; Harrigan, P Richard

2004-01-01

The emergence of drug resistance remains a major problem for the treatment of HIV-infected patients. However, the variety of mutational patterns that evolve in clinical practice have made the application of resistance data to clinical decision-making challenging. Despite (or because of) an abundance of drug-resistance data from disparate sources, there is only limited information available describing the patterns of drug resistance which usually appear in the clinic. Here we attempt to address this issue by reviewing HIV drug resistance in the population of patients failing antiretroviral therapy in British Columbia, Canada from June 1996 to December 2003 as an example. Our findings suggest that, although hundreds of mutations have been associated with resistance, relatively few key mutations occur at a high frequency. For example, only the nucleoside reverse transcriptase inhibitor (NRTI) mutations M184V, M41L T215Y, D67N, K70R and L210W, non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and Y181C, and protease inhibitor (PI) mutation L90M, occur in more than 10% of samples tested for resistance in this population. The introduction of new drugs allows for the selection of new mutations. Trends in the prevalence of resistance-associated mutations have generally followed trends in drug usage, but have not always mirrored them. The phenomenon of cross-resistance can play an important role in the efficacy of new antiretroviral agents, even before they become available. The extent of this cross-resistance depends in part on the prevalence of specific mutations in the population of individuals who have previously received antiretroviral therapy. Hence there is a need to determine which mutations are prevalent in the treated population. The tremendous capacity of HIV to adapt means that common resistance pathways are likely to change over time, and new pathways to resistance are likely to continue to be discovered in the future.

One-, two-, and three-class resistance among HIV-infected patients on antiretroviral therapy in private care clinics: Mumbai, India.

PubMed

Gupta, Amita; Saple, Dattaray G; Nadkarni, Girish; Shah, Bijal; Vaidya, Satish; Hingankar, Nitin; Chaturbhuj, Devidas; Deshmukh, Praveen; Walshe, Louise; Hudelson, Sarah E; James, Maria; Paranjape, Ramesh S; Eshleman, Susan H; Tripathy, Srikanth

2010-01-01

HIV-infected patients receiving antiretroviral (ARV) therapy (ART) in India are not all adequately virally suppressed. We analyzed ARV drug resistance in adults receiving ART in three private clinics in Mumbai, India. HIV viral load was measured in 200 patients with the Roche AMPLICOR HIV-1 Monitor Test, v1.5. HIV genotyping was performed with the ViroSeq HIV-1 Genotyping System for 61 participants who had HIV-1 RNA >1000 copies/ml. Genotyping results were obtained for 51 samples. The participants with resistance results were on ART for a median of 24 months and were on their current regimen for a median of 12 months (median CD4 cell count: 217 cells/mm(3); median HIV viral load: 28,200 copies/ml). ARV regimens included nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (n = 27), dual nucleoside reverse transcriptase inhibitors (NRTIs, n = 19), protease inhibitor (PI)-based regimens (n = 3), and other regimens (n = 2). Twenty-six participants (51.0%) were on their first ARV regimen and 24 (47%) reported >95% adherence. Forty-nine participants (96.1%) had resistance to at least one ARV drug; 47 (92.2%) had NRTI resistance, 32 (62.7%) had NNRTI resistance, and four (7.8%) had PI resistance. Thirty (58.8%) had two-class resistance and three (5.9%) had three-class resistance. Four (8%) had three or more resistance mutations associated with etravirine resistance and two (4%) had two mutations associated with reduced darunavir susceptibility. Almost all patients with HIV-1 RNA >1000 copies/ml had NRTI resistance and nearly two-thirds had NNRTI resistance; PI resistance was uncommon. Nearly 60% and 6% had two- and three-class resistance, respectively. This emphasizes the need for greater viral load and resistance monitoring, use of optimal ART combinations, and increased availability of second- and third-line agents for patients with ARV resistance.

Analysis of drug resistance during HIV RNA viraemia in the MONET trial of darunavir/ritonavir monotherapy.

PubMed

Pulido, Federico; Arribas, José R; Hill, Andrew; Van Delft, Yvon; Moecklinghoff, Christiane

2011-01-01

When patients have HIV RNA suppressed to <50 copies/ml on current treatment, switching to darunavir (DRV)/ritonavir (DRV/r) monotherapy could prevent the development of resistance to other drug classes. In the MONET trial, 256 patients with HIV RNA<50 copies/ml on current highly active antiretroviral therapy (57% with protease inhibitors [PIs] and 43% with non-nucleoside reverse transcriptase inhibitors) and no history of virological failure were randomized to DRV/r 800/100 mg once daily, either as monotherapy (monotherapy arm) or with two nucleoside reverse transcriptase inhibitors (NRTIs; triple therapy arm). All samples with HIV RNA ≥ 50 copies/ml were genotyped, and a virtual phenotype was calculated (VircoType HIV-1 assays; Virco BVBA, Mechelen, Belgium). A total of 63 patients had ≥ 1 HIV RNA result ≥ 50 copies/ml, of whom 38 were successfully genotyped. Most HIV RNA increases were transient and in the range of 50-200 copies/ml. Overall, 36 of the 38 (95%) successfully genotyped patients showed no International AIDS Society-USA major PI mutations, DRV mutations or NRTI mutations. Two patients showed some evidence of PI resistance during transient HIV RNA elevations: one patient in the monotherapy arm had a single DRV mutation (L33F) when HIV RNA was 63 copies/ml (the virus was phenotypically sensitive to DRV [fold change 0.8]) and one PI pretreated patient taking tenofovir disoproxil fumarate/emtricitabine/DRV/r had re-emergence of pre-existing NRTI (M184V) and PI (V82I and L90M) mutations after a short treatment interruption (this virus remained phenotypically sensitive to DRV/r). Both patients showed sustained HIV RNA suppression to week 48 remaining with the same treatment. Emergence of drug resistance after changing a suppressive triple antiretroviral therapy to DRV/r with or without nucleoside analogues is uncommon.

Increase of Transmitted Drug Resistance among HIV-Infected Sub-Saharan Africans Residing in Spain in Contrast to the Native Population

PubMed Central

Yebra, Gonzalo; de Mulder, Miguel; Pérez-Elías, María Jesús; Pérez-Molina, José Antonio; Galán, Juan Carlos; Llenas-García, Jara; Moreno, Santiago; Holguín, África

2011-01-01

Background The prevalence of transmitted HIV drug resistance (TDR) is stabilizing or decreasing in developed countries. However, this trend is not specifically evaluated among immigrants from regions without well-implemented antiretroviral strategies. Methods TDR trends during 1996–2010 were analyzed among naïve HIV-infected patients in Spain, considering their origin and other factors. TDR mutations were defined according to the World Health Organization list. Results Pol sequence was available for 732 HIV-infected patients: 292 native Spanish, 226 sub-Saharan Africans (SSA), 114 Central-South Americans (CSA) and 100 from other regions. Global TDR prevalence was 9.7% (10.6% for Spanish, 8.4% for SSA and 7.9% for CSA). The highest prevalences were found for protease inhibitors (PI) in Spanish (3.1%), for non-nucleoside reverse transcriptase inhibitors (NNRTI) in SSA (6.5%) and for nucleoside reverse transcriptase inhibitors (NRTI) in both Spanish and SSA (6.5%). The global TDR rate decreased from 11.3% in 2004–2006 to 8.4% in 2007–2010. Characteristics related to a decreasing TDR trend in 2007-10 were Spanish and CSA origin, NRTI- and NNRTI-resistance, HIV-1 subtype B, male sex and infection through injection drug use. TDR remained stable for PI-resistance, in patients infected through sexual intercourse and in those carrying non-B variants. However, TDR increased among SSA and females. K103N was the predominant mutation in all groups and periods. Conclusion TDR prevalence tended to decrease among HIV-infected native Spanish and Central-South Americans, but it increased up to 13% in sub-Saharan immigrants in 2007–2010. These results highlight the importance of a specific TDR surveillance among immigrants to prevent future therapeutic failures, especially when administering NNRTIs. PMID:22046345

Time to Switch to Second-line Antiretroviral Therapy in Children With Human Immunodeficiency Virus in Europe and Thailand.

PubMed

2018-02-01

Data on durability of first-line antiretroviral therapy (ART) in children with human immunodeficiency virus (HIV) are limited. We assessed time to switch to second-line therapy in 16 European countries and Thailand. Children aged <18 years initiating combination ART (≥2 nucleoside reverse transcriptase inhibitors [NRTIs] plus nonnucleoside reverse transcriptase inhibitor [NNRTI] or boosted protease inhibitor [PI]) were included. Switch to second-line was defined as (i) change across drug class (PI to NNRTI or vice versa) or within PI class plus change of ≥1 NRTI; (ii) change from single to dual PI; or (iii) addition of a new drug class. Cumulative incidence of switch was calculated with death and loss to follow-up as competing risks. Of 3668 children included, median age at ART initiation was 6.1 (interquartile range (IQR), 1.7-10.5) years. Initial regimens were 32% PI based, 34% nevirapine (NVP) based, and 33% efavirenz based. Median duration of follow-up was 5.4 (IQR, 2.9-8.3) years. Cumulative incidence of switch at 5 years was 21% (95% confidence interval, 20%-23%), with significant regional variations. Median time to switch was 30 (IQR, 16-58) months; two-thirds of switches were related to treatment failure. In multivariable analysis, older age, severe immunosuppression and higher viral load (VL) at ART start, and NVP-based initial regimens were associated with increased risk of switch. One in 5 children switched to a second-line regimen by 5 years of ART, with two-thirds failure related. Advanced HIV, older age, and NVP-based regimens were associated with increased risk of switch. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Recent trends and patterns in HIV-1 transmitted drug resistance in the United Kingdom.

PubMed

Tostevin, A; White, E; Dunn, D; Croxford, S; Delpech, V; Williams, I; Asboe, D; Pozniak, A; Churchill, D; Geretti, A M; Pillay, D; Sabin, C; Leigh-Brown, A; Smit, E

2017-03-01

Transmission of drug-resistant HIV-1 has decreased in the UK since the early 2000s. This analysis reports recent trends and characteristics of transmitted drug resistance (TDR) in the UK from 2010 to 2013. Resistance tests conducted in antiretroviral treatment (ART)-naïve individuals between 2010 and 2013 were analysed for the presence of transmitted drug resistance mutations (TDRMs), defined as any mutations from a modified 2009 World Health Organization surveillance list, or a modified 2013 International Antiviral Society-USA list for integrase tests. Logistic regression was used to examine associations between demographics and the prevalence of TDRMs. TDRMs were observed in 1223 (7.5%) of 16 425 individuals; prevalence declined from 8.1% in 2010 to 6.6% in 2013 (P = 0.02). The prevalence of TDRMs was higher among men who have sex with men (MSM) compared with heterosexual men and women (8.7% versus 6.4%, respectively) with a trend for decreasing TDRMs among MSM (P = 0.008) driven by a reduction in nucleoside reverse transcriptase inhibitor (NRTI)-related mutations. The most frequently detected TDRMs were K103N (2.2%), T215 revertants (1.6%), M41L (0.9%) and L90M (0.7%). Predicted phenotypic resistance to first-line ART was highest to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) rilpivirine and efavirenz (6.2% and 3.4%, respectively) but minimal to NRTIs, including tenofovir, and protease inhibitors (PIs). No major integrase TDRMs were detected among 101 individuals tested while ART-naïve. We observed a decrease in TDRMs in recent years. However, this was confined to the MSM population and rates remained stable in those with heterosexually acquired HIV infection. Resistance to currently recommended first-line ART, including integrase inhibitors, remained reassuringly low. © 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

HIV-1 transmission networks across Cyprus (2010-2012).

PubMed

Kostrikis, Leondios G; Hezka, Johana; Stylianou, Dora C; Kostaki, Evangelia; Andreou, Maria; Kousiappa, Ioanna; Paraskevis, Dimitrios; Demetriades, Ioannis

2018-01-01

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

Transmitted HIV Drug Resistance Is High and Longstanding in Metropolitan Washington, DC

PubMed Central

Kassaye, Seble G.; Grossman, Zehava; Balamane, Maya; Johnston-White, Betsy; Liu, Chenglong; Kumar, Princy; Young, Mary; Sneller, Michael C.; Sereti, Irini; Dewar, Robin; Rehm, Catherine; Meyer, William; Shafer, Robert; Katzenstein, David; Maldarelli, Frank

2016-01-01

Background. Washington, DC, has 2.5% human immunodeficiency virus (HIV) prevalence, 3.9% among African Americans. Antiretrovirals (ARTs) are the cornerstone for treatment and prevention. Monitoring changes in transmitted drug resistance (TDR) is critical for effective HIV care. Methods. HIV genotype data for individuals enrolled in research studies in metropolitan Washington, D.C., were used to identify TDR using the World Health Organization mutation list [Bennett DE, Camacho RJ, Otelea D, et al. Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PloS One 2009; 4:e4724]. HIV phylogenies were reconstructed using maximum likelihood and Bayesian methods. HIV transmission clusters were supported by 1000 bootstrap values >0.70 and posterior probability >0.95 of having a common ancestor. Results. Among 710 individuals enrolled in 1994–2013, the median age was 38.6 years, 46.2% were female, and 53.3% were African-American. TDR was 22.5% among 566 treatment-naive individuals; 15.8% had nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) resistance, 9.8% had nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance, and 4.2% had protease inhibitor (PI) resistance. Single class TDR was 10.0%, 5.1%, and 1.6% to NRTIs, NNRTIs, and PIs. Dual TDR to PI and NRTI was seen in 1.6%, NRTI and NNRTI in 3.4%, and triple class TDR in 0.9%. TDR frequency decreased from 1994–2006 (27.1%) to 2007–2013 (19.4%; P = .02). Only 6/79 (7.6%) individuals within transmission clusters had evidence of TDR. Discussions. We identified high prevalence of TDR among HIV-infected individuals in metropolitan Washington, DC, regardless of gender. Active surveillance for TDR is needed to guide ART usage and analyses of risk group contributions to HIV transmission and resistance. PMID:27307507

Raltegravir plasma concentrations in treatment-experienced patients receiving salvage regimens based on raltegravir with and without maraviroc coadministration.

PubMed

Baroncelli, Silvia; Villani, Paola; Weimer, Liliana E; Ladisa, Nicoletta; Francisci, Daniela; Tommasi, Chiara; Vullo, Vincenzo; Preziosi, Roberta; Cicalini, Stefania; Cusato, Maria; Galluzzo, Clementina; Floridia, Marco; Regazzi, Mario

2010-05-01

Raltegravir and maraviroc represent new, important resources for HIV-infected patients with intolerance or resistance to other antiretroviral agents. The safety and efficacy of both drugs have been investigated, but there is no information on possible pharmacokinetic interactions between these 2 drugs in clinical practice. To evaluate raltegravir plasma concentrations in heavily treatment-experienced patients receiving salvage regimens and explore, in a preliminary assessment, the potential influence of maraviroc coadministration and other cofactors on raltegravir trough concentrations (C(trough)). Fifty-four HIV-infected patients with triple class (nucleoside reverse transcriptase inhibitor, nonnucleoside reverse transcriptase inhibitor, protease inhibitor) treatment experience starting raltegravir 400 mg twice daily, with (n = 11) or without (n = 43) concomitant maraviroc 300 mg twice daily, were evaluated. All regimens included at least 3 drugs of at least 2 different classes. Raltegravir plasma Ctrough, after at least 1 month of treatment, were analyzed to compare groups of patients taking raltegravir only and raltegravir plus maraviroc. Immunovirological (CD4, HIV-RNA) and clinical data after 6 months of treatment were also collected and described. Raltegravir plasma Ctrough showed a large variability (range <0.020-2.47 microg/mL). Median levels were similar in the 2 groups (raltegravir + maraviroc 0.104 microg/mL, range 0.025-0.826; raltegravir 0.090 microg/mL, range <0.020-2.47, p = 0.400). Detectable (>0.02 microg/mL) raltegravir concentrations were observed in all patients receiving raltegravir + maraviroc and in 74% of patients receiving raltegravir alone (p = 0.060). After 6 months of treatment, the 2 groups had similar clinical, virologic, and immunologic conditions. Coadministration of maraviroc does not seem to have any relevant effects on raltegravir plasma Ctrough in heavily treatment-experienced patients receiving salvage regimens. Further studies should evaluate the potential additional benefits of maraviroc coadministration in terms of virologic and immunologic response.

Viro-immunological response of drug-naive HIV-1-infected patients starting a first-line regimen with viraemia >500,000 copies/ml in clinical practice.

PubMed

Santoro, Maria Mercedes; Di Carlo, Domenico; Armenia, Daniele; Zaccarelli, Mauro; Pinnetti, Carmela; Colafigli, Manuela; Prati, Francesca; Boschi, Andrea; Antoni, Anna Maria Degli; Lagi, Filippo; Sighinolfi, Laura; Gervasoni, Cristina; Andreoni, Massimo; Antinori, Andrea; Mussini, Cristina; Perno, Carlo Federico; Borghi, Vanni; Sterrantino, Gaetana

2017-09-22

Virological success (VS) and immunological reconstitution (IR) of antiretroviral-naive HIV-1-infected patients with pre-therapy viral load (VL) >500,000 copies/ml was assessed after 12 months of treatment according to initial drug-class regimens. An observational multicentre retrospective study was performed. VS was defined as the first VL <50 copies/ml from treatment start. IR was defined as an increase of at least 150 CD4 + T-lymphocytes from treatment start. Survival analysis was used to estimate the probability and predictors of VS and IR by 12 months of therapy. 428 HIV-1-infected patients were analysed. Patients were grouped according to the different first-line drug-classes used: a non-nucleoside reverse transcriptase inhibitor (NNRTI) plus two nucleoside reverse transcriptase inhibitors (NRTIs; NNRTI-group; n=105 [24.5%]); a protease inhibitor (PI) plus two NRTIs (PI-group; n=260 [60.8%]); a four-drug regimen containing a PI-regimen plus an integrase inhibitor (PI+INI-group; n=63 [14.7%]). Patients in the PI-group showed the lowest probability of VS (PI-group: 72.4%; NNRTI-group: 75.5%; PI+INI-group: 81.0%; P<0.0001). By Cox regression, patients in PI+INI and NNRTI-groups showed a higher adjusted hazard ratio (95% CI) of VS compared to those in the PI-group (PI+INI-group: 1.48 [1.08, 2.03]; P=0.014; NNRTI-group: 1.37 [1.06-1.78]; P=0.015). The probability of IR was 76.2%, and was similar among groups. Patients with AIDS showed a lower adjusted hazard ratio (95% CI) of IR compared to non-AIDS presenters (0.70 [0.54, 0.90]; P=0.005). In this multicentre retrospective study, patients with viraemia >500,000 copies/ml who start a first-line regimen containing PI+INI or NNRTI yield a better VS compared to those receiving a PI-based regimen.

An assessment of the relationship between the World Health Organization HIV drug resistance early warning indicators and HIV drug resistance acquisition.

PubMed

St-Jean, M; Harrigan, P R; Sereda, P; Montaner, Jsg; Lima, V D

2017-05-01

The World Health Organization (WHO)'s HIV drug resistance (HIVDR) early warning indicators (EWIs) measure antiretroviral therapy (ART)-site factors associated with HIVDR prevention, without HIVDR laboratory testing. We assessed the relationship between EWIs and HIVDR acquisition using data from British Columbia, Canada. Eligible patients were ART-naïve, were ≥ 19 years old, had initiated ART between 1 January 2000 and 31 December 2012, had ≥ 15 months of follow-up, and were without transmitted HIVDR. Patients were followed for acquired HIVDR until 31 March 2014, the last contact date, or death. We built logistic regression models to assess the associations and predictive ability of individual indicators and of the EWI Score (the number of indicators for which a patient did not meet the criteria) on HIVDR acquisition (to any class of HIVDR, lamivudine (3TC)/emtricitabine (FTC), nonnucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs)]). All explored EWIs were associated with at least one class of HIVDR, with the exception of 'ART prescribing practices'. We observed a dose-response relationship between acquiring HIVDR to any antiretroviral class and an increasing EWI score in our predictive logistic regression model. The area under the curve was 0.848 (excellent discrimination). The adjusted odds ratios for acquiring any class of HIVDR for an EWI score of 1, 2 and ≥ 3 versus 0 were 2.30 [95% confidence Interval (CI) 1.21-4.38], 3.35 (95% CI: 1.86-6.03) and 7.26 (95% CI: 4.18-12.61), respectively. Several EWIs were associated with and predictive of HIVDR, supporting the WHO EWIs as a component of the HIVDR prevention method in settings where HIVDR testing is not routinely or widely available. © 2016 British HIV Association.

HIV-1 drug resistance in antiretroviral-naive individuals with HIV-1-associated tuberculous meningitis initiating antiretroviral therapy in Vietnam.

PubMed

Thao, Vu P; Le, Thuy; Török, Estee M; Yen, Nguyen T B; Chau, Tran T H; Jurriaans, Suzanne; van Doorn, H Rogier; van Doorn, Rogier H; de Jong, Menno D; Farrar, Jeremy J; Dunstan, Sarah J

2012-01-01

Access to antiretroviral therapy (ART) for HIV-infected individuals in Vietnam is rapidly expanding, but there are limited data on HIV drug resistance (HIVDR) to guide ART strategies. We retrospectively conducted HIVDR testing in 220 ART-naive individuals recruited to a randomized controlled trial of immediate versus deferred ART in individuals with HIV-associated tuberculous meningitis in Ho Chi Minh City (HCMC) from 2005-2008. HIVDR mutations were identified by population sequencing of the HIV pol gene and were defined based on 2009 WHO surveillance drug resistance mutations (SDRMs). We successfully sequenced 219/220 plasma samples of subjects prior to ART; 218 were subtype CRF01_AE and 1 was subtype B. SDRMs were identified in 14/219 (6.4%) subjects; 8/14 were resistant to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs; T69D, L74V, V75M, M184V/I and K219R), 5/14 to non-nucleoside reverse transcriptase inhibitors (NNRTIs; K103N, V106M, Y181C, Y188C and G190A), 1/14 to both NRTIs and NNRTIs (D67N and Y181C) and none to protease inhibitors. After 6 months of ART, eight subjects developed protocol-defined virological failure. HIVDR mutations were identified in 5/8 subjects. All five had mutations with high-level resistance to NNRTIs and three had mutations with high-level resistance to NRTIs. Due to a high early mortality rate (58%), the effect of pre-existing HIVDR mutations on treatment outcome could not be accurately assessed. The prevalence of WHO SDRMs in ART-naive individuals with HIV-associated tuberculous meningitis in HCMC from 2005-2008 is 6.4%. The SDRMs identified conferred resistance to NRTIs and/or NNRTIs, reflecting the standard first-line ART regimens in Vietnam.

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

PubMed Central

Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

2008-01-01

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

Reverse Transcriptase Activity in Mature Spermatozoa of Mouse

PubMed Central

Giordano, Roberto; Magnano, Anna Rosa; Zaccagnini, Germana; Pittoggi, Carmine; Moscufo, Nicola; Lorenzini, Rodolfo; Spadafora, Corrado

2000-01-01

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development. PMID:10725323

Unconventional plasticity of HIV-1 reverse transcriptase: how inhibitors could open a connection "gate" between allosteric and catalytic sites.

PubMed

Bellucci, Luca; Angeli, Lucilla; Tafi, Andrea; Radi, Marco; Botta, Maurizio

2013-12-23

Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection "gate" between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors.

In Vitro Evaluation of Nonnucleoside Reverse Transcriptase Inhibitors UC-781 and TMC120-R147681 as Human Immunodeficiency Virus Microbicides†

PubMed Central

Van Herrewege, Yven; Michiels, Jo; Van Roey, Jens; Fransen, Katrien; Kestens, Luc; Balzarini, Jan; Lewi, Paul; Vanham, Guido; Janssen, Paul

2004-01-01

The nonnucleoside reverse transcriptase inhibitors UC-781 and TMC120-R147681 (Dapivirine) effectively prevented human immunodeficiency virus (HIV) infection in cocultures of monocyte-derived dendritic cells and T cells, representing primary targets in sexual transmission. Both drugs had a favorable therapeutic index. A 24-h treatment with 1,000 nM UC-781 or 100 nM TMC120-R147681 prevented cell-free HIV infection, whereas 10-fold-higher concentrations blocked cell-associated HIV. PMID:14693562

Gamma-irradiated bacterial preparation having anti-tumor activity

DOEpatents

Vass, Arpad A.; Tyndall, Richard L.; Terzaghi-Howe, Peggy

1999-01-01

A bacterial preparation from Pseudomonas species isolated #15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

PubMed

Atwood, A; Lin, J H; Levin, H L

1996-01-01

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Characteristics of a group of nonnucleoside reverse transcriptase inhibitors with structural diversity and potent anti-human immunodeficiency virus activity.

PubMed

Yang, S S; Fliakas-Boltz, V; Bader, J P; Buckheit, R W

1995-10-01

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

Efavirenz or nevirapine in three-drug combination therapy with two nucleoside-reverse transcriptase inhibitors for initial treatment of HIV infection in antiretroviral-naïve individuals.

PubMed

Mbuagbaw, Lawrence Ce; Irlam, James H; Spaulding, Alicen; Rutherford, George W; Siegfried, Nandi

2010-12-08

The advent of highly active antiretroviral therapy (HAART) has reduced the morbidity and mortality due to HIV. The World Health Organisation (WHO) antiretroviral treatment (ART) guidelines focus on three classes of antiretroviral drugs, namely: nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Two of the most common medications given in first-line treatment are the NNRTIs, efavirenz (EFV) and nevirapine (NVP). It is unclear which NNRTI is more efficacious for initial therapy. To determine which NNRTI, EFV or NVP, is more efficacious when given in combination with two NRTIs as part of initial ART for HIV infection in adults and children. We used a comprehensive and exhaustive strategy in an attempt to identify all relevant studies, regardless of language or publication status, in electronic databases and conference proceedings from 1996 to 2009. All randomised controlled trials comparing EFV to NVP in HIV-infected individuals without prior exposure to ART, irrespective of the dosage or NRTI backbone.The primary outcome of interest was virologic response to ART. Other primary outcomes included mortality, clinical progression, severe adverse events, and discontinuation of therapy for any reason. Secondary outcomes were immunologic response to ART, treatment failure, development of ART drug resistance, and prevention of sexual transmission of HIV. Two authors assessed each reference for inclusion and exclusion criteria established a priori. Data were abstracted independently using a standardised abstraction form. Data were analysed on an intention-to-treat basis and reported as per dosage of NVP. We identified seven randomised controlled trials that met our inclusion criteria.The trials were pooled as per dosage of NVP. None of these trials included children.The seven trials enrolled 1,688 participants and found no critical differences between EFV and NVP, except for different toxicity profiles. EFV is more likely to cause central nervous system side-effects, while NVP is more likely to result in raised transaminases and neutropoenia. There was a higher mortality rate in the NVP 400mg once daily arm.The quality of literature to support these conclusions is moderate to high. Drug resistance was slightly less common with EFV than NVP, but the quality of this literature is low since only one of the seven studies reported on this outcome. No studies reported on sexual transmission of HIV. The length of follow-up time, study settings, and NRTI backbone varied greatly. Both drugs have equivalent efficacies in initial treatment of HIV infection when combined with two NRTIs, but different side effects.

High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

PubMed Central

Cancio, Reynel; Silvestri, Romano; Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Crespan, Emmanuele; Zanoli, Samantha; Hübscher, Ulrich; Spadari, Silvio; Maga, Giovanni

2005-01-01

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation. PMID:16251294

Synthesis, Activity and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Chung, Suhman; Himmel, Daniel M.; Jiang, Jian-Kang; Wojtak, Krzysztof; Bauman, Joseph D.; Rausch, Jason W.; Wilson, Jennifer A.; Beutler, John A.; Thomas, Craig J.; Arnold, Eddy; Le Grice, Stuart F.J.

2011-01-01

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use. PMID:21568335

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Pelliccia, Sveva; Brancale, Andrea; Maga, Giovanni; Crespan, Emmanuele; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Ferretti, Rosella; Cirilli, Roberto; Zamperini, Claudio; Botta, Maurizio; Schols, Dominique; Limongelli, Vittorio; Agostino, Bruno; Novellino, Ettore; Silvestri, Romano

2014-12-11

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

Structure-based virtual screening efforts against HIV-1 reverse transcriptase to introduce the new potent non-nucleoside reverse transcriptase inhibitor

NASA Astrophysics Data System (ADS)

Hosseini, Yaser; Mollica, Adriano; Mirzaie, Sako

2016-12-01

The human immunodeficiency virus (HIV) which is strictly related to the development of AIDS, is treated by a cocktail of drugs, but due its high propensity gain drug resistance, the rational development of new medicine is highly desired. Among the different mechanism of action we selected the reverse transcriptase (RT) inhibition, for our studies. With the aim to identify new chemical entities to be used for further rational drug design, a set of 3000 molecules from the Zinc Database have been screened by docking experiments using AutoDock Vina software. The best ranked compounds with respect of the crystallographic inhibitor MK-4965 resulted to be five compounds, and the best among them was further tested by molecular dynamics (MD) simulation. Our results indicate that comp1 has a stronger interaction with the subsite p66 of RT than MK-4965 and that both are able to stabilize specific conformational changes of the RT 3D structure, which may explain their activity as inhibitors. Therefore comp1 could be a good candidate for biological tests and further development.

Studies on the inhibition of Moloney murine leukemia virus reverse transcriptase by N-tritylamino acids and N-tritylamino acid-nucleotide compounds.

PubMed

Hawtrey, Arthur; Pieterse, Anton; van Zyl, Johann; Van der Bijl, Pieter; Van der Merwe, Marichen; Nel, William; Ariatti, Mario

2008-09-01

N-Acylated derivatives of 8-(6-aminohexyl) amino-adenosine-5 '-phosphate were prepared and studied with regard to their effect on DNA synthesis by the Moloney leukemia virus reverse transcriptase. N-palmitoyl and N-nicotinyl derivatives and bis-8-(6-aminohexyl) amino-5'-AMP inhibited the enzyme partially using poly (rA).oligo d(pT)(16-18) as template-primer with [(3)H]dTTP. In order to increase hydrophobicity in the acyl component tethered to the 8-(6-aminohexyl) amino group on the adenine nucleotide, N-trityl-L-phenylalanine and the N-trityl derivatives of the o, m, and p-fluoro-DL-phenylalanine were initially examined for inhibition of the enzyme using the above template-primer system. The compounds all inhibited the reverse transcriptase with IC(50) values of approximately 60-80 microM. However, when N-trityl-m-fluoro-DL-phenylalanine was coupled to the nucleotide 8-(6-aminohexyl) amino-adenosine-5'-phosphate, the inhibitory activity of this compound increased significantly (IC(50) = 5 microM).

Polyurethane intravaginal ring for controlled delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1.

PubMed

Gupta, Kavita M; Pearce, Serena M; Poursaid, Azadeh E; Aliyar, Hyder A; Tresco, Patrick A; Mitchnik, Mark A; Kiser, Patrick F

2008-10-01

Women-controlled methods for prevention of male-to-female sexual transmission of HIV-1 are urgently needed. Providing inhibitory concentrations of HIV-1 reverse transcriptase inhibitors to impede the replication of the virus in the female genital tissue offers a mechanism for prophylaxis of HIV-1. To this end, an intravaginal ring device that can provide long duration delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1, was developed utilizing a medical-grade polyether urethane. Monolithic intravaginal rings were fabricated and sustained release with cumulative flux linear with time was demonstrated under sink conditions for a period of 30 days. The release rate was directly proportional to the amount of drug loaded. Another release study conducted for a week utilizing liposome dispersions as sink conditions, to mimic the partitioning of dapivirine into vaginal tissue, also demonstrated release rates constant with time. These results qualify polyether urethanes for development of intravaginal rings for sustained delivery of microbicidal agents. (c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Combination antiretroviral use and preterm birth.

PubMed

Watts, D Heather; Williams, Paige L; Kacanek, Deborah; Griner, Raymond; Rich, Kenneth; Hazra, Rohan; Mofenson, Lynne M; Mendez, Hermann A

2013-02-15

Use of antiretroviral drugs (ARVs) during pregnancy has been associated with higher risk of preterm birth. The Pediatric HIV/AIDS Cohort Study network's Surveillance Monitoring for ART Toxicities study is a US-based cohort of human immunodeficiency virus (HIV)-exposed uninfected children. We evaluated maternal ARV use during pregnancy and the risk of any type of preterm birth (ie, birth before 37 completed weeks of gestation), the risk of spontaneous preterm birth (ie, preterm birth that occurred after preterm labor or membrane rupture, without other complications), and the risk of small for gestational age (SGA; ie, a birth weight of <10th percentile for gestational age). Multivariable logistic regression models were used to evaluate the association of ARVs and timing of exposure, while adjusting for maternal characteristics. Among 1869 singleton births, 18.6% were preterm, 10.2% were spontaneous preterm, and 7.3% were SGA. A total of 89% used 3-drug combination ARV regimens during pregnancy. In adjusted models, the odds of preterm birth and spontaneous preterm birth were significantly greater among mothers who used protease inhibitors during the first trimester (adjusted odds ratios, 1.55 and 1.59, respectively) but not among mothers who used nonnucleoside reverse-transcriptase inhibitor or triple-nucleoside regimens during the first trimester. Combination ARV exposure starting later in pregnancy was not associated with increased risk. No associations were observed between SGA and exposure to combination ARV regimens. Protease inhibitor use early in pregnancy may be associated with increased risk for prematurity.

Gamma-irradiated bacterial preparation having anti-tumor activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vass, A.A.; Tyndall, R.L.; Terzaghi-Howe, P.

1999-11-16

This application describes a bacterial preparation from Pseudomonas species isolated {number{underscore}sign}15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Tenofovir-related nephrotoxicity: case report and review of the literature.

PubMed

James, Christopher W; Steinhaus, Mary C; Szabo, Susan; Dressier, Robert M

2004-03-01

Tenofovir is a nucleotide reverse transcriptase inhibitor for treatment of human immunodeficiency virus (HIV) infection. Several cases of renal failure associated with tenofovir therapy recently have been reported. A 54-year-old man with HIV experienced decreasing renal function and Fanconi's syndrome secondary to tenofovir therapy. His condition gradually improved after discontinuation of the drug. The available medical literature for reported cases of tenofovir-related nephrotoxicity indicates that this complication is apparently rare. However, our case report and literature review underscore the importance of monitoring renal function when treating patients with any nucleotide reverse transcriptase inhibitor.

Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, Biological Activities and Mechanism of Action

PubMed Central

Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Artico, Marino; Lavecchia, Antonio; Marinelli, Luciana; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

2008-01-01

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the β-diketo acids (DKAs) represent a major lead for anti-HIV-1drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-crosslinking experiments. PMID:16539381

Dyslipidemia in a Cohort of HIV-infected Latin American Children Receiving Highly Active Antiretroviral Therapy*

PubMed Central

Brewinski, Margaret; Megazzini, Karen; Freimanis Hance, Laura; Cruz, Miguel Cashat; Pavia-Ruz, Noris; Della Negra, Marinella; Ferreira, Flavia Gomes Faleiro; Marques, Heloisa

2011-01-01

In order to describe the prevalence of hypercholesterolemia and hypertriglyceridemia in a cohort of HIV-infected children and adolescents in Latin America and to determine associations with highly active antiretroviral therapy (HAART), we performed this cross-sectional analysis within the NICHD International Site Development Initiative pediatric cohort study. Eligible children had to be at least 2 years of age and be on HAART. Among the 477 eligible HIV-infected youth, 98 (20.5%) had hypercholesterolemia and 140 (29.4%) had hypertriglyceridemia. In multivariable analyses, children receiving protease inhibitor (PI)-containing HAART were at increased risk for hypercholesterolemia [adjusted odds ratio (AOR) = 2.7, 95% confidence interval (CI) 1.3–5.6] and hypertriglyceridemia (AOR = 3.5, 95% CI 1.9–6.4) compared with children receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing HAART. In conclusion, HIV-infected youth receiving PI-containing HAART in this Latin American cohort were at increased risk for hypercholesterolemia and hypertriglyceridemia compared with those receiving NNRTI-containing HAART. PMID:20889625

The biochemistry and medical significance of the flavonoids.

PubMed

Havsteen, Bent H

2002-01-01

Flavonoids are plant pigments that are synthesised from phenylalanine, generally display marvelous colors known from flower petals, mostly emit brilliant fluorescence when they are excited by UV light, and are ubiquitous to green plant cells. The flavonoids are used by botanists for taxonomical classification. They regulate plant growth by inhibition of the exocytosis of the auxin indolyl acetic acid, as well as by induction of gene expression, and they influence other biological cells in numerous ways. Flavonoids inhibit or kill many bacterial strains, inhibit important viral enzymes, such as reverse transcriptase and protease, and destroy some pathogenic protozoans. Yet, their toxicity to animal cells is low. Flavonoids are major functional components of many herbal and insect preparations for medical use, e.g., propolis (bee's glue) and honey, which have been used since ancient times. The daily intake of flavonoids with normal food, especially fruit and vegetables, is 1-2 g. Modern authorised physicians are increasing their use of pure flavonoids to treat many important common diseases, due to their proven ability to inhibit specific enzymes, to simulate some hormones and neurotransmitters, and to scavenge free radicals.

Fanconi syndrome induced by tenofovir: A case report.

PubMed

Lify, Bouchra; Dabo, G; Nascimento, O; Iraqui, S; Elkhayat, S; Zamd, M; Medkouri, G; Benghanem, M; Ramdani, B; Sodqi, M M; Marih, L; Chakib, A; El FilaliMarhoum, K

2016-01-01

Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor discovered in the USA in 2001. It is currently the treatment of choice for patients co-infected with human immunodeficiency virus (HIV) and hepatitis B virus. Its antiretroviral efficacy and good tolerance are responsible for the higher frequency of prescriptions compared with other nucleoside analogs. However, it can induce acute renal toxicity causing impairment of the proximal tubular function of the kidney. This is highly dependent on factors such as associated co-prescription didanosine or a protease inhibitor "boosted" with ritonavir, preexisting renal insufficiency, low body weight, or presence of associated diabetes. In contrast, long-term renal toxicity remains highly debated. Some studies describe a decrease in estimated glomerular filtration rate during prolonged treatment with TDF. Others reported renal safety even during prolonged use. The differences between patients enrolled in the different studies, the measured parameters and their interpretation could explain these discrepancies. We describe a case of a patient infected with HIV, who presented with Fanconi syndrome with acute renal failure six months after starting antiretroviral treatment including tenofovir.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Therapy-Naive Individuals Residing in Sorong, West Papua.

PubMed

Witaningrum, Adiana Mutamsari; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Yunifiar M, Muhammad Qushai; Indriati, Dwi Wahyu; Bramanthi, Rendra; Nasronudin; Kameoka, Masanori

2016-08-01

Papua and West Papua provinces have the highest prevalence rate of human immunodeficiency virus type 1 (HIV-1) infection in Indonesia; however, data on the molecular epidemiology of HIV-1 are limited. We conducted a genotypic study on HIV-1 genes derived from antiretroviral therapy-naive individuals residing in Sorong, West Papua. HIV-1 genomic fragments were amplified from 43 peripheral blood samples, and sequencing analysis of the genes was carried out. Of the 43 samples, 41 protease (PR), 31 reverse transcriptase (RT), 26 gag, and 25 env genes were sequenced. HIV-1 subtyping revealed that CRF01_AE (48.8%, 21/43) and subtype B (41.9%, 18/43) were the major subtypes prevalent in the region, whereas other recombinant forms were also detected. Major drug resistance-associated mutations for PR inhibitors were not detected; however, mutations for the RT inhibitors, A62V and E138A, appeared in a few samples, indicating the possible emergence of transmitted HIV-1 drug resistance in Sorong, West Papua.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Drug-Treated Individuals Residing in Earthquake-Affected Areas in Nepal.

PubMed

Negi, Bharat Singh; Kotaki, Tomohiro; Joshi, Sunil Kumar; Bastola, Anup; Nakazawa, Minato; Kameoka, Masanori

2017-09-01

Molecular epidemiological data on human immunodeficiency virus type 1 (HIV-1) are limited in Nepal and have not been available in areas affected by the April 2015 earthquake. Therefore, we conducted a genotypic study on HIV-1 genes derived from individuals on antiretroviral therapy residing in 14 districts in Nepal highly affected by the earthquake. HIV-1 genomic fragments were amplified from 40 blood samples of HIV treatment-failure individuals, and a sequencing analysis was performed on these genes. In the 40 samples, 29 protease, 32 reverse transcriptase, 25 gag, and 21 env genes were sequenced. HIV-1 subtyping revealed that subtype C (84.2%, 32/38) was the major subtype prevalent in the region, while CRF01_AE (7.9%, 3/38) and other recombinant forms (7.9%, 3/38) were also detected. In addition, major drug resistance mutations were identified in 21.9% (7/32) of samples, indicating the possible emergence of HIV-1 drug resistance in earthquake-affected areas in Nepal.

Demonstration of retrotransposition of the Tf1 element in fission yeast.

PubMed

Levin, H L; Boeke, J D

1992-03-01

Tf1, a retrotransposon from fission yeast, has LTRs and coding sequences resembling the protease, reverse transcriptase and integrase domains of retroviral pol genes. A unique aspect of Tf1 is that it contains a single open reading frame whereas other retroviruses and retrotransposons usually possess two or more open reading frames. To determine whether Tf1 can transpose, we overproduced Tf1 transcripts encoded by a plasmid copy of the element marked with a neo gene. Approximately 0.1-4.0% of the cell population acquired chromosomally inherited resistance to G418. DNA blot analysis demonstrated that such strains had acquired both Tf1 and neo specific sequences within a restriction fragment of the same size; the size of this restriction fragment varied between different isolates. Structural analysis of the cloned DNA flanking the Tf1-neo element of two transposition candidates with the same regions in the parent strain showed that the ability to grow on G418 was due to transposition of Tf1-neo and not other types of recombination events.

Antiretroviral drug resistance and phylogenetic diversity of HIV-1 in Chile.

PubMed

Ríos, Maritza; Delgado, Elena; Pérez-Alvarez, Lucía; Fernández, Jorge; Gálvez, Paula; de Parga, Elena Vázquez; Yung, Verónica; Thomson, Michael M; Nájera, Rafael

2007-06-01

This study reports the analysis of human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) coding sequences from 136 HIV-1-infected subjects from Chile, 66 (49%) of them under antiretroviral (ARV) treatment. The prevalence of mutations conferring high or intermediate resistance levels to ARVs was 77% among treated patients and 2.5% among drug-naïve subjects. The distribution of resistance prevalence in treated patients by drug class was 61% to nucleoside RT inhibitors, 84% to nonnucleoside RT inhibitors, and 46% to PR inhibitors. Phylogenetic analysis revealed that 115 (85%) subjects were infected with subtype B viruses, 1 with a subtype F1 virus, and 20 (15%) carried BF intersubtype recombinants. Most BF recombinants grouped into two clusters, one related to CRF12_BF, while the other could represent a new circulating recombinant form (CRF). In conclusion, this is the first report analysing the prevalence of ARV resistance which includes patients under HAART from Chile. Additionally, phylogenetic analysis of the PR-RT coding sequences reveals the presence of BF intersubtype recombinants. (c) 2007 Wiley-Liss, Inc.

High HIV-1 Diversity and Prevalence of Transmitted Drug Resistance Among Antiretroviral-Naive HIV-Infected Pregnant Women from Rio de Janeiro, Brazil.

PubMed

Delatorre, Edson; Silva-de-Jesus, Carlos; Couto-Fernandez, José Carlos; Pilotto, Jose H; Morgado, Mariza G

2017-01-01

Antiretroviral (ARV) resistance mutations in human immunodeficiency virus type 1 (HIV-1) infection may reduce the efficacy of prophylactic therapy to prevent mother-to-child transmission (PMTCT) and future treatment options. This study evaluated the diversity and the prevalence of transmitted drug resistance (TDR) in protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene among 87 ARV-naive HIV-1-infected pregnant women from Rio de Janeiro, Brazil, between 2012 and 2015. The viral diversity comprised HIV-1 subtypes B (67.8%), F1 (17.2%), and C (4.6%); the circulating recombinant forms 12_BF (2.3%), 28/29_BF, 39_BF, 02_AG (1.1% each) and unique recombinants forms (4.5%). The overall prevalence of any TDR was 17.2%, of which 5.7% for nucleoside RT inhibitors, 5.7% for non-nucleoside RT inhibitors, and 8% for PR inhibitors. The TDR prevalence found in this population may affect the virological outcome of the standard PMTCT ARV-regimens, reinforcing the importance of continuous monitoring.

Computational challenges of structure-based approaches applied to HIV.

PubMed

Forli, Stefano; Olson, Arthur J

2015-01-01

Here, we review some of the opportunities and challenges that we face in computational modeling of HIV therapeutic targets and structural biology, both in terms of methodology development and structure-based drug design (SBDD). Computational methods have provided fundamental support to HIV research since the initial structural studies, helping to unravel details of HIV biology. Computational models have proved to be a powerful tool to analyze and understand the impact of mutations and to overcome their structural and functional influence in drug resistance. With the availability of structural data, in silico experiments have been instrumental in exploiting and improving interactions between drugs and viral targets, such as HIV protease, reverse transcriptase, and integrase. Issues such as viral target dynamics and mutational variability, as well as the role of water and estimates of binding free energy in characterizing ligand interactions, are areas of active computational research. Ever-increasing computational resources and theoretical and algorithmic advances have played a significant role in progress to date, and we envision a continually expanding role for computational methods in our understanding of HIV biology and SBDD in the future.

Resistance Analyses of Integrase Strand Transfer Inhibitors within Phase 3 Clinical Trials of Treatment-Naive Patients

PubMed Central

White, Kirsten L.; Raffi, Francois; Miller, Michael D.

2014-01-01

The integrase (IN) strand transfer inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG), comprise the newest drug class approved for the treatment of HIV-1 infection, which joins the existing classes of reverse transcriptase, protease and binding/entry inhibitors. The efficacy of first-line regimens has attained remarkably high levels, reaching undetectable viral loads in 90% of patients by Week 48; however, there remain patients who require a change in regimen due to adverse events, virologic failure with emergent resistance or other issues of patient management. Large, randomized clinical trials conducted in antiretroviral treatment-naive individuals are required for drug approval in this population in the US, EU and other countries, with the primary endpoint for virologic success at Week 48. However, there are differences in the definition of virologic failure and the evaluation of drug resistance among the trials. This review focuses on the methodology and tabulation of resistance to INSTIs in phase 3 clinical trials of first-line regimens and discusses case studies of resistance. PMID:25054884

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

PubMed

Verweij-van Wissen, C P W G M; Aarnoutse, R E; Burger, D M

2005-02-25

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro.

PubMed

Ortega, Joseph T; Suárez, Alirica I; Serrano, Maria L; Baptista, Jani; Pujol, Flor H; Rangel, Hector R

2017-10-12

Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns

PubMed Central

Mohr, Georg; Ghanem, Eman; Lambowitz, Alan M.

2010-01-01

Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelments hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting ∼1.3% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases. PMID:20543989

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

PubMed

Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Early antiretroviral therapy: rationale, protease inhibitor-sparing regimens and once daily dosing.

PubMed

Gatell, J M

1998-01-01

In 1998 it seems reasonable and widely accepted that all human immunodeficiency virus type 1 (HIV-1)-infected patients willing to be treated may benefit from receiving antiretroviral therapy. Only those with undetectable plasma HIV-1 RNA, normal CD4 lymphocyte counts and lack of markers of immunological system activation may be possible exceptions. The rationale supporting the early initiation of antiretroviral therapy are (i) data on viral dynamics; (ii) preliminary data pointing toward a better and a quicker restoration of immune function when treatment is initiated in very early stages (during or within a few weeks or months of acute symptomatic or asymptomatic HIV-1 infection); (iii) the lack of a stable viral load set-point even in patients in the early stages (CD4 > 500 cells/mm3) who have a very low viral load (< 5000 copies/ml); (iv) the relatively high likelihood of clinical progression at mid-term of the approximately 50-75% of patients in very early disease stages (CD4 > 500 cells/mm3) who have a plasma viral load above 5000 to 10,000 HIV-1 RNA copies/ml; (v) data from the Spanish Earth-1 study, which used a composite endpoint (virological, immunological or clinical progression), demonstrating that even in these very early stages of HIV-1 disease any antiretroviral therapy (double or triple combination) was better than no treatment. Even in early disease stages, a triple combination is needed to achieve a durable and profound virological and immunological response. In addition, the combination of stavudine plus didanosine has several advantages and can be considered one of the best double nucleoside combinations to combine with a protease inhibitor or with a non-nucleoside reverse transcriptase inhibitor. The INCAS study and the preliminary results of the ongoing Spanish SCAN study have demonstrated the possibility of protease inhibitor-sparing combinations for initial antiretroviral treatment, at least in selected patient subsets, such as those with a relatively low baseline viral load. Furthermore, components of these protease inhibitor-sparing combinations such as didanosine or nevirapine are suitable for once daily administration.

Quantitative Assessment of the Sensitivity of Various Commercial Reverse Transcriptases Based on Armored HIV RNA

PubMed Central

Okello, John B. A.; Rodriguez, Linda; Poinar, Debi; Bos, Kirsten; Okwi, Andrew L.; Bimenya, Gabriel S.; Sewankambo, Nelson K.; Henry, Kenneth R.; Kuch, Melanie; Poinar, Hendrik N.

2010-01-01

Background The in-vitro reverse transcription of RNA to its complementary DNA, catalyzed by the enzyme reverse transcriptase, is the most fundamental step in the quantitative RNA detection in genomic studies. As such, this step should be as analytically sensitive, efficient and reproducible as possible, especially when dealing with degraded or low copy RNA samples. While there are many reverse transcriptases in the market, all claiming to be highly sensitive, there is need for a systematic independent comparison of their applicability in quantification of rare RNA transcripts or low copy RNA, such as those obtained from archival tissues. Methodology/Principal Findings We performed RT-qPCR to assess the sensitivity and reproducibility of 11 commercially available reverse transcriptases in cDNA synthesis from low copy number RNA levels. As target RNA, we used a serially known number of Armored HIV RNA molecules, and observed that 9 enzymes we tested were consistently sensitive to ∼1,000 copies, seven of which were sensitive to ∼100 copies, while only 5 were sensitive to ∼10 RNA template copies across all replicates tested. Despite their demonstrated sensitivity, these five best performing enzymes (Accuscript, HIV-RT, M-MLV, Superscript III and Thermoscript) showed considerable variation in their reproducibility as well as their overall amplification efficiency. Accuscript and Superscript III were the most sensitive and consistent within runs, with Accuscript and Superscript II ranking as the most reproducible enzymes between assays. Conclusions/Significance We therefore recommend the use of Accuscript or Superscript III when dealing with low copy number RNA levels, and suggest purification of the RT reactions prior to downstream applications (eg qPCR) to augment detection. Although the results presented in this study were based on a viral RNA surrogate, and applied to nucleic acid lysates derived from archival formalin-fixed paraffin embedded tissue, their relative performance on RNA obtained from other tissue types may vary, and needs future evaluation. PMID:21085668

Lentin, a novel and potent antifungal protein from shitake mushroom with inhibitory effects on activity of human immunodeficiency virus-1 reverse transcriptase and proliferation of leukemia cells.

PubMed

Ngai, Patrick H K; Ng, T B

2003-11-14

From the fruiting bodies of the edible mushroom Lentinus edodes, a novel protein designated lentin with potent antifungal activity was isolated. Lentin was unadsorbed on DEAE-cellulose, and adsorbed on Affi-gel blue gel and Mono S. The N-terminal sequence of lentin manifested similarity to endoglucanase. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

Structure-Based Design of Novel Dihydroalkoxybenzyloxopyrimidine Derivatives as Potent Nonnucleoside Inhibitors of the Human Immunodeficiency Virus Reverse Transcriptase

PubMed Central

Sudbeck, Elise A.; Mao, Chen; Vig, Rakesh; Venkatachalam, T. K.; Tuel-Ahlgren, Lisa; Uckun, Fatih M.

1998-01-01

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 μM. PMID:9835518

HIV Resistance Prediction to Reverse Transcriptase Inhibitors: Focus on Open Data.

PubMed

Tarasova, Olga; Poroikov, Vladimir

2018-04-19

Research and development of new antiretroviral agents are in great demand due to issues with safety and efficacy of the antiretroviral drugs. HIV reverse transcriptase (RT) is an important target for HIV treatment. RT inhibitors targeting early stages of the virus-host interaction are of great interest for researchers. There are a lot of clinical and biochemical data on relationships between the occurring of the single point mutations and their combinations in the pol gene of HIV and resistance of the particular variants of HIV to nucleoside and non-nucleoside reverse transcriptase inhibitors. The experimental data stored in the databases of HIV sequences can be used for development of methods that are able to predict HIV resistance based on amino acid or nucleotide sequences. The data on HIV sequences resistance can be further used for (1) development of new antiretroviral agents with high potential for HIV inhibition and elimination and (2) optimization of antiretroviral therapy. In our communication, we focus on the data on the RT sequences and HIV resistance, which are available on the Internet. The experimental methods, which are applied to produce the data on HIV-1 resistance, the known data on their concordance, are also discussed.

Chemical system biology based molecular interactions to identify inhibitors against Q151M mutant of HIV-1 reverse transcriptase.

PubMed

Pandey, Rajan Kumar; Sharma, Drista; Ojha, Rupal; Bhatt, Tarun Kumar; Prajapati, Vijay Kumar

2018-05-09

The emergence of mutations leading to drug resistance is the main cause of therapeutic failure in the human HIV infection. Chemical system biology approach has drawn great attention to discover new antiretroviral hits with high efficacy and negligible toxicity, which can be used as a prerequisite for HIV drug resistance global action plan 2017-21. To discover potential hits, we docked 49 antiretroviral analogs (n = 6294) against HIV-1 reverse transcriptase Q151M mutant & its wild-type form and narrow downed their number in three sequential modes of docking using Schrödinger suite. Later on, 80 ligands having better docking score than reference ligands (tenofovir and lamivudine) were screened for ADME, toxicity prediction, and binding energy estimation. Simultaneously, the area under the curve (AUC) was estimated using receiver operating characteristics (ROC) curve analysis to validate docking protocols. Finally, single point energy and molecular dynamics simulation approaches were performed for best two ligands (L3 and L14). This study reveals the antiretroviral efficacy of obtained two best ligands and delivers the hits against HIV-1 reverse transcriptase Q151M mutant. Copyright © 2018 Elsevier B.V. All rights reserved.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

PubMed

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-17

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase

PubMed Central

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-01

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth. This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors. PMID:27926505

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed Central

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-01-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay. PMID:7512096

Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

PubMed Central

Debyser, Z.; De Clercq, E.

1996-01-01

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

PubMed

Chang, G J; Trent, D W; Vorndam, A V; Vergne, E; Kinney, R M; Mitchell, C J

1994-02-01

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Structure-based methods to predict mutational resistance to diarylpyrimidine non-nucleoside reverse transcriptase inhibitors.

PubMed

Azeem, Syeda Maryam; Muwonge, Alecia N; Thakkar, Nehaben; Lam, Kristina W; Frey, Kathleen M

2018-01-01

Resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a leading cause of HIV treatment failure. Often included in antiviral therapy, NNRTIs are chemically diverse compounds that bind an allosteric pocket of enzyme target reverse transcriptase (RT). Several new NNRTIs incorporate flexibility in order to compensate for lost interactions with amino acid conferring mutations in RT. Unfortunately, even successful inhibitors such as diarylpyrimidine (DAPY) inhibitor rilpivirine are affected by mutations in RT that confer resistance. In order to aid drug design efforts, it would be efficient and cost effective to pre-evaluate NNRTI compounds in development using a structure-based computational approach. As proof of concept, we applied a residue scan and molecular dynamics strategy using RT crystal structures to predict mutations that confer resistance to DAPYs rilpivirine, etravirine, and investigational microbicide dapivirine. Our predictive values, changes in affinity and stability, are correlative with fold-resistance data for several RT mutants. Consistent with previous studies, mutation K101P is predicted to confer high-level resistance to DAPYs. These findings were further validated using structural analysis, molecular dynamics, and an enzymatic reverse transcription assay. Our results confirm that changes in affinity and stability for mutant complexes are predictive parameters of resistance as validated by experimental and clinical data. In future work, we believe that this computational approach may be useful to predict resistance mutations for inhibitors in development. Published by Elsevier Inc.

Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

PubMed

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L; Bertagnolio, Silvia; Rinke de Wit, Tobias F; Aghokeng, Avelin F; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O; Brooks, James I; Boucher, Charles A B; Brumme, Zabrina L; Busch, Michael P; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T; De Gascun, Cillian F; Derache, Anne; Descamps, Diane; Deshpande, Alaka K; Djoko, Cyrille F; Eshleman, Susan H; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S; Peeters, Martine; Poljak, Mario; Price, Matt A; Ragonnet-Cronin, Manon L; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M; Soares, Marcelo A; Soriano, Vincent V; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M; van de Vijver, David A M C; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P A; Vandamme, Anne-Mieke; Shafer, Robert W

2015-04-01

Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

PubMed Central

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R.; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L.; Bertagnolio, Silvia; de Wit, Tobias F. Rinke; Aghokeng, Avelin F.; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O.; Brooks, James I.; Boucher, Charles A. B.; Brumme, Zabrina L.; Busch, Michael P.; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D’Aquin, Toni T.; De Gascun, Cillian F.; Derache, Anne; Descamps, Diane; Deshpande, Alaka K.; Djoko, Cyrille F.; Eshleman, Susan H.; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P. Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M.; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H.; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; NG, Kee Peng; Paranjape, Ramesh S.; Peeters, Martine; Poljak, Mario; Price, Matt A.; Ragonnet-Cronin, Manon L.; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M.; Soares, Marcelo A.; Soriano, Vincent V.; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A.; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M.; van de Vijver, David A. M. C.; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P. A.; Vandamme, Anne-Mieke; Shafer, Robert W.

2015-01-01

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen. PMID:25849352

copia-like retrotransposons are ubiquitous among plants.

PubMed Central

Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

1992-01-01

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

The evolution of HIV-1 group M genetic variability in Southern Cameroon is characterized by several emerging recombinant forms of CRF02_AG and viruses with drug resistance mutations.

PubMed

Agyingi, Lucy; Mayr, Luzia M; Kinge, Thompson; Orock, George Enow; Ngai, Johnson; Asaah, Bladine; Mpoame, Mbida; Hewlett, Indira; Nyambi, Phillipe

2014-03-01

The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by circulating recombinant forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naïve patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naïve patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naïve patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1. © 2013 Wiley Periodicals, Inc.

Combination Antiretroviral Use and Preterm Birth

PubMed Central

Watts, D. Heather; Williams, Paige L.; Kacanek, Deborah; Griner, Raymond; Rich, Kenneth; Hazra, Rohan; Mofenson, Lynne M.; Mendez, Hermann A.

2013-01-01

Background. Use of antiretroviral drugs (ARVs) during pregnancy has been associated with higher risk of preterm birth. Methods. The Pediatric HIV/AIDS Cohort Study network's Surveillance Monitoring for ART Toxicities study is a US-based cohort of human immunodeficiency virus (HIV)–exposed uninfected children. We evaluated maternal ARV use during pregnancy and the risk of any type of preterm birth (ie, birth before 37 completed weeks of gestation), the risk of spontaneous preterm birth (ie, preterm birth that occurred after preterm labor or membrane rupture, without other complications), and the risk of small for gestational age (SGA; ie, a birth weight of <10th percentile for gestational age). Multivariable logistic regression models were used to evaluate the association of ARVs and timing of exposure, while adjusting for maternal characteristics. Results. Among 1869 singleton births, 18.6% were preterm, 10.2% were spontaneous preterm, and 7.3% were SGA. A total of 89% used 3-drug combination ARV regimens during pregnancy. In adjusted models, the odds of preterm birth and spontaneous preterm birth were significantly greater among mothers who used protease inhibitors during the first trimester (adjusted odds ratios, 1.55 and 1.59, respectively) but not among mothers who used nonnucleoside reverse-transcriptase inhibitor or triple-nucleoside regimens during the first trimester. Combination ARV exposure starting later in pregnancy was not associated with increased risk. No associations were observed between SGA and exposure to combination ARV regimens. Conclusions. Protease inhibitor use early in pregnancy may be associated with increased risk for prematurity. PMID:23204173

[Genetic analysis of the mutations in HIV-1 infected population in Ecuador].

PubMed

González-González, Manuel; Correa-Sierra, Consuelo; Hermida-Álava, Katherine; Machado-Díaz, Ana; Gómez-Andrade, L Fernando; Castillo-Segovia, Martha; Pérez-Santos, C Lissette; Kourí-Cardellá, Vivian

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

HIV-1 Amino Acid Changes Among Participants With Virologic Failure: Associations With First-line Efavirenz or Atazanavir Plus Ritonavir and Disease Status

PubMed Central

Mollan, Katie; Daar, Eric S.; Sax, Paul E.; Balamane, Maya; Collier, Ann C.; Fischl, Margaret A.; Lalama, Christina M.; Bosch, Ronald J.; Tierney, Camlin; Katzenstein, David

2012-01-01

Background. Although specific human immunodeficiency virus type 1 (HIV-1) drug resistance mutations are well studied, little is known about cumulative amino acid changes, or how regimen and participant characteristics influence these changes. Methods. In the AIDS Clinical Trials Group randomized study A5202 of treatment-naive HIV-infected participants, cumulative HIV-1 amino acid changes from pretreatment to virologic failure were evaluated in protease and reverse transcriptase (RT) gene sequences. Results. Among 265 participants with virologic failure, those assigned atazanavir plus ritonavir (ATV/r) did not have significantly more protease changes compared with those assigned efavirenz (EFV) (P ≥ .13). In contrast, participants with virologic failure assigned EFV had more RT changes, including and excluding known resistance codons (P < .001). At pretreatment, lower CD4 cell count, major resistance, more amino acid mixtures (all P < .001), hepatitis C antibody negativity (P = .05), and black race/ethnicity (P = .02) were associated with more HIV-1 amino acid changes. Conclusions. Virologic failure following EFV-containing treatment was associated with more HIV-1 amino acid changes compared to failure of ATV/r-containing treatment. Furthermore, we show that non–drug resistance mutations occurred more frequently among those failing EFV, the clinical relevance of which warrants further investigation. Pretreatment immunologic status may play a role in viral evolution during treatment, as evidenced by increased amino acid changes among those with lower pretreatment CD4 count. Clinical Trials Registration. NCT00118898. PMID:23148287

[Thyroid dysfunction in adults infected by human immunodeficiency virus].

PubMed

Abelleira, Erika; De Cross, Graciela A; Pitoia, Fabián

2014-01-01

Patients infected with human immunodeficiency virus (HIV) have a higher prevalence of thyroid dysfunction when compared with the general population. The most frequently observed manifestations are euthyroid sick syndrome, Graves' disease and subclinical hypothyroidism. The relationship between the use of highly active antiretroviral therapy and the increased prevalence of thyroid dysfunction has been demonstrated in several series of patients. Grave's disease is recognized as a consequence of immune restitution syndrome. Besides, several studies have suggested an association between hypothyroidism and the use of nucleoside reverse transcriptase inhibitors, particularly stavudine and non-nucleoside reverse transcriptase inhibitors such as efavirenz. Further studies could provide additional evidence of the need for routine assessment of thyroid function in HIV-infected patients.

In search of a treatment for HIV--current therapies and the role of non-nucleoside reverse transcriptase inhibitors (NNRTIs).

PubMed

Reynolds, Chevonne; de Koning, Charles B; Pelly, Stephen C; van Otterlo, Willem A L; Bode, Moira L

2012-07-07

The human immunodeficiency virus (HIV) causes AIDS (acquired immune deficiency syndrome), a disease in which the immune system progressively deteriorates, making sufferers vulnerable to all manner of opportunistic infections. Currently, world-wide there are estimated to be 34 million people living with HIV, with the vast majority of these living in sub-Saharan Africa. Therefore, an important research focus is development of new drugs that can be used in the treatment of HIV/AIDS. This review gives an overview of the disease and addresses the drugs currently used for treatment, with specific emphasis on new developments within the class of allosteric non-nucleoside reverse transcriptase inhibitors (NNRTIs).

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

PubMed Central

Hu, J; Seeger, C

1996-01-01

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577714

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection

PubMed Central

Giacobbi, Nicholas S.

2017-01-01

ABSTRACT Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. PMID:28507107

Endothelial Function in HIV-Infected Antiretroviral Naïve Subjects Before and After Starting Potent Antiretroviral Therapy: AIDS Clinical Trials Group Study 5152s

PubMed Central

Torriani, Francesca J.; Komarow, Lauren; Parker, Robert A.; Cotter, Bruno R.; Currier, Judith S.; Dubé, Michael P.; Fichtenbaum, Carl J.; Gerschenson, Mariana; Mitchell, Carol K.C.; Murphy, Robert L.; Squires, Kathleen; Stein, James H.

2008-01-01

Objectives This study evaluated the effects of three class-sparing antiretroviral therapy (ART) regimens on endothelial function in HIV-infected subjects participating in a randomized trial. Background Endothelial dysfunction has been observed in patients receiving ART for human immunodeficiency virus (HIV) infection. Methods This was a prospective, multicenter study of treatment-naïve subjects who were randomly assigned to receive a protease inhibitor-sparing regimen of nucleoside reverse transcriptase inhibitors (NRTIs) + efavirenz, a non-nucleoside reverse transcriptase inhibitor-sparing regimen of NRTIs + lopinavir/ritonavir, or a NRTI-sparing regimen of efavirenz + lopinavir/ritonavir. NRTIs were lamivudine + stavudine, zidovudine, or tenofovir. Brachial artery flow-mediated dilation (FMD) was determined by B-mode ultrasound before starting on ART, then after 4 and 24 weeks. Results There were 82 subjects (median age 35 years, 91% men, 54% white). Baseline CD4 cell counts and plasma HIV RNA values were 245 cells/mm3 and 4.8 log10 copies/ml, respectively. At baseline, FMD was 3.68% (interquartile range 1.98 – 5.51%). After 4 and 24 weeks of ART, plasma HIV RNA decreased by 2.1 and 3.0 log10 copies/mL, respectively. FMD increased by 0.74% (−0.62 – +2.74, p=0.003) and 1.48% (−0.20 – +4.30%, p< 0.001), respectively, with similar changes in each arm (pKW>0.600). The decrease in plasma HIV RNA at 24 weeks was associated with greater FMD (rs=− 0.30, p=0.017). Conclusions Among treatment-naïve individuals with HIV, three different ART regimens rapidly improved endothelial function. Benefits were similar for all ART regimens, appeared quickly, and persisted at 24 weeks. Condensed Abstract Among 82 treatment-naïve HIV-infected subjects participating in a prospective, multicenter study of three class-sparing antiretroviral therapy regimens, flow-mediated dilation of the brachial artery improved after 4 (+0.74%, p=0.003) and 24 weeks (+1.48%, p< 0.001), with similar changes in each arm (pKW>0.600). PMID:18687253

Mutation covariation of HIV-1 CRF07_BC reverse transcriptase during antiretroviral therapy.

PubMed

Li, Zhenpeng; Huang, Yang; Ouyang, Yabo; Xing, Hui; Liao, Lingjie; Jiang, Shibo; Shao, Yiming; Ma, Liying

2013-11-01

To understand the effect of HIV-1 drug resistance mutations in the context of antiretroviral therapy (ART), we compared the prevalence of protease (PR) and reverse transcriptase (RT) mutations in HIV-1 CRF07_BC sequences from blood samples of treatment-naive and ART-treated patients. Mutation covariation in the RT and PR of HIV-1 CRF07_BC viruses from 542 treatment-naive patients and 261 patients treated with lamivudine/zidovudine/nevirapine or lamivudine/zidovudine/efavirenz was analysed. Stratified networks were used to display the mutation covariation. Based on the comparison between treatment-naive and ART-treated patients, three types of featured mutations for RT and PR were initially identified: treatment-associated mutations, treatment-agonistic mutations and overlapping polymorphisms. Twelve significant covariation pairs were found between five treatment-associated mutations (K103N, M184V, Q197K, G190A and Y181C) and nine overlapping polymorphisms (A36E, D39N, Y121H, D123E, R135I, T200A, R277K, L283I and D291E). Meanwhile, three covariation pairs between three treatment-associated mutations (I132L and M184V for RT and I15V for PR) and three overlapping polymorphisms (L10I, L36M and A71V) for PR were also detected. Finally, the overlapping polymorphisms for RT and PR were both found to have significant correlations with treatment-associated mutations, indicating a possible association between polymorphisms and drug resistance. When compared with HIV-1 subtype B under the same regimens as CRF07_BC, the mutation covariations of CRF07_BC showed a distinct pattern of RT and PR mutation covariation. The role of polymorphisms in the development of drug resistance has been widely reported. Here, we found a significant correlation between overlapping polymorphisms for RT and PR and treatment-associated mutations, indicating that polymorphisms exert a global influence on treatment-associated mutations. Polymorphism mutations might therefore be considered before initiating ART to improve the efficacy of drug combinations.

HIV-1 Drug Resistance and Second-Line Treatment in Children Randomized to Switch at Low Versus Higher RNA Thresholds.

PubMed

Harrison, Linda; Melvin, Ann; Fiscus, Susan; Saidi, Yacine; Nastouli, Eleni; Harper, Lynda; Compagnucci, Alexandra; Babiker, Abdel; McKinney, Ross; Gibb, Diana; Tudor-Williams, Gareth

2015-09-01

The PENPACT-1 trial compared virologic thresholds to determine when to switch to second-line antiretroviral therapy (ART). Using PENPACT-1 data, we aimed to describe HIV-1 drug resistance accumulation on first-line ART by virologic threshold. PENPACT-1 had a 2 × 2 factorial design, randomizing HIV-infected children to start protease inhibitor (PI) versus nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART, and switch at a 1000 copies/mL versus 30,000 copies/mL threshold. Switch criteria were not achieving the threshold by week 24, confirmed rebound above the threshold thereafter, or Center for Disease Control and Prevention stage C event. Resistance tests were performed on samples ≥1000 copies/mL before switch, resuppression, and at 4-years/trial end. Sixty-seven children started PI-based ART and were randomized to switch at 1000 copies/mL (PI-1000), 64 PIs and 30,000 copies/mL (PI-30,000), 67 NNRTIs and 1000 copies/mL (NNRTI-1000), and 65 NNRTI and 30,000 copies/mL (NNRTI-30,000). Ninety-four (36%) children reached the 1000 copies/mL switch criteria during 5-year follow-up. In 30,000 copies/mL threshold arms, median time from 1000 to 30,000 copies/mL switch criteria was 58 (PI) versus 80 (NNRTI) weeks (P = 0.81). In NNRTI-30,000, more nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations accumulated than other groups. NNRTI mutations were selected before switching at 1000 copies/mL (23% NNRTI-1000, 27% NNRTI-30,000). Sixty-two children started abacavir + lamivudine, 166 lamivudine + zidovudine or stavudine, and 35 other NRTIs. The abacavir + lamivudine group acquired fewest NRTI mutations. Of 60 switched to second-line, 79% PI-1000, 63% PI-30,000, 64% NNRTI-1000, and 100% NNRTI-30,000 were <400 copies/mL 24 weeks later. Children on first-line NNRTI-based ART who were randomized to switch at a higher virologic threshold developed the most resistance, yet resuppressed on second-line. An abacavir + lamivudine NRTI combination seemed protective against development of NRTI resistance.

Abacavir–Lamivudine versus Tenofovir–Emtricitabine for Initial HIV-1 Therapy

PubMed Central

Sax, Paul E.; Tierney, Camlin; Collier, Ann C.; Fischl, Margaret A.; Mollan, Katie; Peeples, Lynne; Godfrey, Catherine; Jahed, Nasreen C.; Myers, Laurie; Katzenstein, David; Farajallah, Awny; Rooney, James F.; Ha, Belinda; Woodward, William C.; Koletar, Susan L.; Johnson, Victoria A.; Geiseler, P. Jan; Daar, Eric S.

2009-01-01

BACKGROUND The use of fixed-dose combination nucleoside reverse-transcriptase inhibitors (NRTIs) with a nonnucleoside reverse-transcriptase inhibitor or a ritonavir-boosted protease inhibitor is recommended as initial therapy in patients with human immunodeficiency virus type 1 (HIV-1) infection, but which NRTI combination has greater efficacy and safety is not known. METHODS In a randomized, blinded equivalence study involving 1858 eligible patients, we compared four once-daily anti retroviral regimens as initial therapy for HIV-1 infection: abacavir–lamivudine or tenofovir disoproxil fumarate (DF)–emtricitabine plus efavirenz or ritonavir-boosted atazanavir. The primary efficacy end point was the time from randomization to virologic failure (defined as a confirmed HIV-1 RNA level ≥1000 copies per milliliter at or after 16 weeks and before 24 weeks, or ≥200 copies per milliliter at or after 24 weeks). RESULTS A scheduled interim review by an independent data and safety monitoring board showed significant differences in virologic efficacy, according to the NRTI combination, among patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more. At a median follow-up of 60 weeks, among the 797 patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more, the time to virologic failure was significantly shorter in the abacavir–lamivudine group than in the tenofovir DF–emtricitabine group (hazard ratio, 2.33; 95% confidence interval, 1.46 to 3.72; P<0.001), with 57 virologic failures (14%) in the abacavir–lamivudine group versus 26 (7%) in the tenofovir DF–emtricitabine group. The time to the first adverse event was also shorter in the abacavir–lamivudine group (P<0.001). There was no significant difference between the study groups in the change from the baseline CD4 cell count at week 48. CONCLUSIONS In patients with screening HIV-1 RNA levels of 100,000 copies per milliliter or more, the times to virologic failure and the first adverse event were both significantly shorter in patients randomly assigned to abacavir–lamivudine than in those assigned to tenofovir DF–emtricitabine. (ClinicalTrials.gov number, NCT00118898.) PMID:19952143

HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

PubMed

de Mulder, M; York, V A; Wiznia, A A; Michaud, H A; Nixon, D F; Holguin, A; Rosenberg, M G

2014-03-01

With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/μL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (< 50 copies/mL) and 40.5% had a CD4 count of > 500 cells/μL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection. © 2013 British HIV Association.

Perinatal acquisition of drug-resistant HIV-1 infection: mechanisms and long-term outcome

PubMed Central

Delaugerre, Constance; Chaix, Marie-Laure; Blanche, Stephane; Warszawski, Josiane; Cornet, Dorine; Dollfus, Catherine; Schneider, Veronique; Burgard, Marianne; Faye, Albert; Mandelbrot, Laurent; Tubiana, Roland; Rouzioux, Christine

2009-01-01

Background Primary-HIV-1-infection in newborns that occurs under antiretroviral prophylaxis that is a high risk of drug-resistance acquisition. We examine the frequency and the mechanisms of resistance acquisition at the time of infection in newborns. Patients and Methods We studied HIV-1-infected infants born between 01 January 1997 and 31 December 2004 and enrolled in the ANRS-EPF cohort. HIV-1-RNA and HIV-1-DNA samples obtained perinatally from the newborn and mother were subjected to population-based and clonal analyses of drug resistance. If positive, serial samples were obtained from the child for resistance testing. Results Ninety-two HIV-1-infected infants were born during the study period. Samples were obtained from 32 mother-child pairs and from another 28 newborns. Drug resistance was detected in 12 newborns (20%): drug resistance to nucleoside reverse transcriptase inhibitors was seen in 10 cases, non-nucleoside reverse transcriptase inhibitors in two cases, and protease inhibitors in one case. For 9 children, the detection of the same resistance mutations in mothers' samples (6 among 10 available) and in newborn lymphocytes (6/8) suggests that the newborn was initially infected by a drug-resistant strain. Resistance variants were either transmitted from mother-to-child or selected during subsequent temporal exposure under suboptimal perinatal prophylaxis. Follow-up studies of the infants showed that the resistance pattern remained stable over time, regardless of antiretroviral therapy, suggesting the early cellular archiving of resistant viruses. The absence of resistance in the mother of the other three children (3/10) and neonatal lymphocytes (2/8) suggests that the newborns were infected by a wild-type strain without long-term persistence of resistance when suboptimal prophylaxis was stopped. Conclusion This study confirms the importance of early resistance genotyping of HIV-1-infected newborns. In most cases (75%), drug resistance was archived in the cellular reservoir and persisted during infancy, with or without antiretroviral treatment. This finding stresses the need for effective antiretroviral treatment of pregnant women. PMID:19765313

Genotypic tropism testing of proviral DNA to guide maraviroc initiation in aviraemic subjects: 48-week analysis of results from the PROTEST study.

PubMed

Poveda, E; Hernández-Quero, J; Pérez-Elías, M J; Ribas, M A; Martínez-Madrid, O J; Flores, J; Navarro, J; Gutiérrez, F; García-Deltoro, M; Imaz, A; Ocampo, A; Artero, A; Blanco, F; Bernal, E; Pasquau, J; Mínguez-Gallego, C; Pérez, N; Aiestaran, A; García, F; Paredes, R

2017-08-01

Maraviroc (MVC) is a suitable drug for aviraemic subjects on antiretroviral treatment (ART) developing toxicity. Its prescription requires prior tropism testing. It is unknown if proviral DNA genotypic tropism testing is reliable for guiding MVC initiation in aviraemic subjects, so this study was carried out to address this issue. PROTEST was a phase 4, prospective, single-arm clinical trial carried out in 24 HIV care centres in Spain. MVC-naïve HIV-1-infected patients with HIV-1 RNA < 50 copies/mL on stable ART during the previous 6 months who required an ART change because of toxicity and who had R5 HIV, as determined by proviral DNA genotypic tropism testing, initiated MVC with two nucleoside reverse transcriptase inhibitors (NRTIs) and were followed for 48 weeks. Virological failure was defined as two consecutive viral load measurements > 50 copies/mL. Tropism results were available for 141 of 175 (80.6%) subjects screened: 60% had R5 and 85% of these (n = 74) were finally included in the study. Previous ART included protease inhibitors (PIs) in 62% of subjects, nonnucleoside reverse transcriptase inhibitors (NNRTIs) in 36%, and integrase inhibitors (INIs) in 2%. Main reasons for treatment change were dyslipidaemia (42%), gastrointestinal symptoms (22%) and liver toxicity (15%). MVC was given alongside tenofovir (TDF)/emtricitabine (FTC) (54%) and abacavir (ABC)/lamivudine (3TC) (40%) in most patients. Eighty-four per cent of patients maintained a viral load < 50 copies/mL to week 48, whereas 16% discontinued treatment: two withdrew informed consent, one had an R5 to X4 shift between screening and baseline, one was lost to follow-up, one developed an adverse event (rash), two died from non-study-related causes, and five developed protocol-defined virological failure. Initiation of MVC plus two NRTIs in aviraemic subjects based on genotypic tropism testing of proviral HIV-1 DNA is associated with low rates of virological failure for up to 1 year. © 2016 British HIV Association.

Trends in Transmission of Drug Resistance and Prevalence of Non-B Subtypes in Patients with Acute or Recent HIV-1 Infection in Barcelona in the Last 16 Years (1997-2012).

PubMed

Ambrosioni, Juan; Sued, Omar; Nicolas, David; Parera, Marta; López-Diéguez, María; Romero, Anabel; Agüero, Fernando; Marcos, María Ángeles; Manzardo, Christian; Zamora, Laura; Gómez-Carrillo, Manuel; Gatell, José María; Pumarola, Tomás; Miró, José María

2015-01-01

To evaluate the prevalence of transmitted drug resistance (TDR) and non-B subtypes in patients with acute/recent HIV-1 infection in Barcelona during the period 1997-2012. Patients from the "Hospital Clínic Primary HIV-1 Infection Cohort" with a genotyping test performed within 180 days of infection were included. The 2009 WHO List of Mutations for Surveillance of Transmitted HIV-1 Drug Resistance was used for estimating the prevalence of TDR and phylogenetic analysis for subtype determination. 189 patients with acute/recent HIV-1 infection were analyzed in 4 time periods (1997-2000, n=28; 2001-4, n=42; 2005-8, n=55 and 2009-12, n=64). The proportion of patients with acute/recent HIV-1 infection with respect to the total of newly HIV-diagnosed patients in our center increased over the time and was 2.18%, 3.82%, 4.15% and 4.55% for the 4 periods, respectively (p=0.005). The global prevalence of TDR was 9%, or 17.9%, 9.5%, 3.6% and 9.4% by study period (p=0.2). The increase in the last period was driven by protease-inhibitor and nucleoside-reverse-transcriptase-inhibitor resistance mutations while non-nucleoside-reverse-transcriptase inhibitor TDR and TDR of more than one family decreased. The overall prevalence of non-B subtypes was 11.1%, or 0%, 4.8%, 9.1% and 20.3 by study period (p=0.01). B/F recombinants, B/G recombinants and subtype F emerged in the last period. We also noticed an increase in the number of immigrant patients (p=0.052). The proportion of men-who-have-sex-with-men (MSM) among patients with acute/recent HIV-1 infection increased over the time (p=0.04). The overall prevalence of TDR in patients with acute/recent HIV-1 infection in Barcelona was 9%, and it has stayed relatively stable in recent years. Non-B subtypes and immigrants proportions progressively increased.

Switching regimens in virologically suppressed HIV-1-infected patients: evidence base and rationale for integrase strand transfer inhibitor (INSTI)-containing regimens.

PubMed

Raffi, F; Esser, S; Nunnari, G; Pérez-Valero, I; Waters, L

2016-10-01

In an era when most individuals with treated HIV infection can expect to live into old age, clinicians should proactively review their patients' current and future treatment needs and challenges. Clinical guidelines acknowledge that, in the setting of virological suppression, treatment switch may yield benefits in terms of tolerability, regimen simplification, adherence, convenience and long-term health considerations, particularly in the context of ageing. In this paper, we review evidence from six key clinical studies on switching virologically suppressed patients to regimens based on integrase strand transfer inhibitors (INSTIs), the antiretroviral class increasingly preferred as initial therapy in clinical guidelines. We review these studies and focus on the virological efficacy, safety, and tolerability of switching to INSTI-based regimens in suppressed HIV-positive individuals. We review the early switch studies SWITCHMRK and SPIRAL [assessing a switch from a ritonavir-boosted protease inhibitor (PI/r) to raltegravir (RAL)-containing regimens], together with data from STRATEGY-PI [assessing a switch to elvitegravir (EVG)-containing regimens; EVG/cobicistat (COBI)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) vs. remaining on a PI/r-containing regimen], STRATEGY-NNRTI [assessing a switch to EVG/COBI/FTC/TDF vs. continuation of a nonnucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTIs)], STRIIVING [assessing a switch to a dolutegravir (DTG)-containing regimen (abacavir (ABC)/lamivudine (3TC)/DTG) vs. staying on the background regimen], and GS study 109 [assessing a switch to EVG/COBI/FTC/tenofovir alafenamide fumarate (TAF) vs. continuation of FTC/TDF-based regimens]. Switching to INSTI-containing regimens has been shown to support good virological efficacy, with evidence from two studies demonstrating superior virological efficacy for a switch to EVG-containing regimens. In addition, switching to INSTI regimens was associated with improved tolerability and greater reported patient satisfaction and outcomes in some studies. INSTI-based regimens offer an important contemporary switch option that may be tailored to meet and optimize the needs of many patients. © 2016 British HIV Association.

An integrated molecular dynamics, principal component analysis and residue interaction network approach reveals the impact of M184V mutation on HIV reverse transcriptase resistance to lamivudine.

PubMed

Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

2014-08-01

The emergence of different drug resistant strains of HIV-1 reverse transcriptase (HIV RT) remains of prime interest in relation to viral pathogenesis as well as drug development. Amongst those mutations, M184V was found to cause a complete loss of ligand fitness. In this study, we report the first account of the molecular impact of M184V mutation on HIV RT resistance to 3TC (lamivudine) using an integrated computational approach. This involved molecular dynamics simulation, binding free energy analysis, principle component analysis (PCA) and residue interaction networks (RINs). Results clearly confirmed that M184V mutation leads to steric conflict between 3TC and the beta branched side chain of valine, decreases the ligand (3TC) binding affinity by ∼7 kcal mol(-1) when compared to the wild type, changes the overall conformational landscape of the protein and distorts the native enzyme residue-residue interaction network. The comprehensive molecular insight gained from this study should be of great importance in understanding drug resistance against HIV RT as well as assisting in the design of novel reverse transcriptase inhibitors with high ligand efficacy on resistant strains.

Dipyridodiazepinone analogs as human immunodeficiency virus type 1-specific non-nucleoside reverse transcriptase inhibitors: an overview.

PubMed

Lv, M; Xu, H

2010-01-01

According to World Health Organization (WHO)/Joint United Nations Programme on human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) (UNAIDS) Report in 2007, 33.2 million people are living with HIV, 2.5 million ones have been newly infected with HIV, and 2.1 million ones died from AIDS, including 330,000 children. Therefore, HIV/AIDS still remains a public health emergency and a leading cause of mortality worldwide. It is believed that reverse transcriptase (RT) is a crucial enzyme in the life cycle of HIV-1, and thereby RT has been the important drug target for antiretroviral (ARV) chemotherapy against AIDS. To our knowledge, dipyridodiazepinone analogs have been considered as one class of potential non-nucleoside reverse transcriptase inhibitors (NNRTIs), especially the structurally and chemically related nevirapine (Viramune(R)), which was the first NNRTI approved by the U. S. Food and Drug Administration (FDA) for the treatment of HIV-1 infection for adults in 1996 and for children in 1998. This review mainly highlights the progress of synthesis and structure-activity relationship (SAR) of dipyridodiazepinone analogs; in the meantime, the mechanism of action is also presented. It will pave the way for the design and development of novel dipyridodiazepinone analogs as NNRTIs in AIDS chemotherapy in the future.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

Occurrence of transmitted HIV-1 drug resistance among Drug-naïve pregnant women in selected HIV-care centres in Ghana.

PubMed

Martin-Odoom, Alexander; Adiku, Theophilus; Delgado, Elena; Lartey, Margaret; Ampofo, William K

2017-03-01

Access to antiretroviral therapy in Ghana has been scaled up across the country over the last decade. This study sought to determine the occurrence of transmitted HIV-1 drug resistance in pregnant HIV-1 positive women yet to initiate antiretroviral therapy at selected HIV Care Centres in Ghana. Plasma specimens from twenty-six (26) HIV seropositive pregnant women who were less than 28weeks pregnant with their first pregnancy and ART naïve were collected from selected HIV care centres in three (3) regions in Ghana. Genotypic testing was done for the reverse transcriptase gene and the sequences generated were analyzed for HIV-1 drug resistance mutations using the Stanford University HIV Drug Resistance Database. Resistance mutations associated with the reverse transcriptase gene were detected in 4 (15.4%) of the participants. At least one major drug resistance mutation in the reverse transcriptase gene was found in 3 (11.5%) of the women. The detection of transmitted HIV-1 drug resistance in this drug-naïve group in two regional HIV care sites is an indication of the need for renewed action in monitoring the emergence of transmitted HIV-1 drug resistance in Ghana. None declared.

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

PubMed Central

Zajac, Pawel; Islam, Saiful; Hochgerner, Hannah; Lönnerberg, Peter; Linnarsson, Sten

2013-01-01

Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells. PMID:24392002

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

PubMed Central

Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

2016-01-01

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from <1 ng of plasma RNA in <5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

[The use of complex interval models for predicting activity of non-nucleoside reverse transcriptase activity].

PubMed

Burliaeva, E V; Tarkhov, A E; Burliaev, V V; Iurkevich, A M; Shvets, V I

2002-01-01

Searching of new anti-HIV agents is still crucial now. In general, researches are looking for inhibitors of certain HIV's vital enzymes, especially for reverse transcriptase (RT) inhibitors. Modern generation of anti-HIV agents represents non-nucleoside reverse transcriptase inhibitors (NNRTIs). They are much less toxic than nucleoside analogues and more chemically stable, thus being slower metabolized and emitted from the human body. Thus, search of new NNRTIs is actual today. Synthesis and study of new anti-HIV drugs is very expensive. So employment of the activity prediction techniques for such a search is very beneficial. This technique allows predicting the activities for newly proposed structures. It is based on the property model built by investigation of a series of known compounds with measured activity. This paper presents an approach of activity prediction based on "structure-activity" models designed to form a hypothesis about probably activity interval estimate. This hypothesis formed is based on structure descriptor domains, calculated for all energetically allowed conformers for each compound in the studied sef. Tetrahydroimidazobenzodiazipenone (TIBO) derivatives and phenylethyltiazolyltiourea (PETT) derivatives illustrated the predictive power of this method. The results are consistent with experimental data and allow to predict inhibitory activity of compounds, which were not included into the training set.

Susceptibility of recombinant porcine endogenous retrovirus reverse transcriptase to nucleoside and non-nucleoside inhibitors.

PubMed

Wilhelm, M; Fishman, J A; Pontikis, R; Aubertin, A M; Wilhelm, F X

2002-12-01

Transplantation of organs, tissues or cells from pigs to humans could be a potential solution to the shortage of human organs for transplantation. Porcine endogenous retroviruses (PERVs) remain a major safety concern for porcine xenotransplantation. Thus, finding drugs that could be used as virological prophylaxis (or therapy) against PERV replication would be desirable. One of the most effective ways to block retroviral multiplication is to inhibit the enzyme reverse transcriptase (RT) which catalyzes the reverse transcription of viral RNA to proviral double-stranded DNA. We report here the cloning and expression of PERV RT and its susceptibility to several inhibitors. Our data demonstrate PERV susceptibility in vitro to the triphosphorylated nucleoside analog of zidovudine (AZT) and to ddGTP and to a lesser extent to ddTTP but almost no susceptibility to the non-nucleoside RT inhibitors tested.

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site,more » confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.« less

Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors

PubMed Central

Xu, Hong-Tao; Colby-Germinario, Susan P.; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J.

2013-01-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV. PMID:24002090

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J; Wainberg, Mark A

2013-11-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Maturation inhibitors: a new therapeutic class targets the virus structure.

PubMed

Salzwedel, Karl; Martin, David E; Sakalian, Michael

2007-01-01

The current standard of care for HIV/AIDS in the developed world is HAART therapy, usually a combination of two reverse transcriptase inhibitors and a protease inhibitor. Despite the success of this regimen, there is a continuing need for new drug options to overcome problems with tolerability and the emergence of viral resistance. In this review we discuss the discovery of a potential new class of antiretroviral therapeutics, known as maturation inhibitors, and the development of the first-in-class compound, bevirimat. Bevirimat is distinguished from the currently available antiretrovirals by its unique target and mode of action. While the specific interactions responsible for activity have yet to be fully characterized, it is clear that the target for bevirimat is the Gag polyprotein precursor, the main structural protein responsible for assembly and budding of virion particles. As basic research continues on the precise mechanism of action of bevirimat, clinical development is progressing, with demonstration of both safety and efficacy in early-stage trials. These encouraging results, coupled with the discovery and development of future generations of maturation inhibitors, suggest that maturation inhibitors may be added to the growing set of tools available to control HIV/AIDS.

Biology and clinical relevance of chemokines and chemokine receptors CXCR4 and CCR5 in human diseases

PubMed Central

Choi, Won-Tak; An, Jing

2014-01-01

Chemokines and their receptors are implicated in a wide range of human diseases, including acquired immune deficiency syndrome (AIDS). The entry of human immunodeficiency virus type 1 (HIV-1) into a cell is initiated by the interaction of the virus’s surface envelope proteins with two cell surface components of the target cell, namely CD4 and a chemokine co-receptor, usually CXCR4 or CCR5. Typical anti-HIV-1 agents include protease and reverse transcriptase inhibitors, but the targets of these agents tend to show rapid mutation rates. As such, strategies based on HIV-1 co-receptors have appeal because they target invariant host determinants. Chemokines and their receptors are also of general interest since they play important roles in numerous physiological and pathological processes in addition to AIDS. Therefore, intensive basic and translational research is ongoing for the dissection of their structure – function relationships in an effort to understand the molecular mechanism of chemokine – receptor interactions and signal transductions across cellular membranes. This paper reviews and discusses recent advances and the translation of new knowledge and discoveries into novel interventional strategies for clinical application. PMID:21565895

Gene signature based on degradome-related genes can predict distal metastasis in cervical cancer patients.

PubMed

Fernandez-Retana, Jorge; Zamudio-Meza, Horacio; Rodriguez-Morales, Miguel; Pedroza-Torres, Abraham; Isla-Ortiz, David; Herrera, Luis; Jacobo-Herrera, Nadia; Peralta-Zaragoza, Oscar; López-Camarillo, César; Morales-Gonzalez, Fermin; Cantu de Leon, David; Pérez-Plasencia, Carlos

2017-06-01

Cervical cancer is one of the leading causes of death in women worldwide, which mainly affects developing countries. The patients who suffer a recurrence and/or progression disease have a higher risk of developing distal metastases. Proteases comprising the degradome given its ability to promote cell growth, migration, and invasion of tissues play an important role during tumor development and progression. In this study, we used high-density microarrays and quantitative reverse transcriptase polymerase chain reaction to evaluate the degradome profile and their inhibitors in 112 samples of patients diagnosed with locally advanced cervical cancer. Clinical follow-up was done during a period of 3 years. Using a correlation analysis between the response to treatment and the development of metastasis, we established a molecular signature comprising eight degradome-related genes (FAM111B, FAM111A, CFB, PSMB8, PSMB9, CASP7, PRSS16, and CD74) with the ability to discriminate patients at risk of distal metastases. In conclusion, present results show that molecular signature obtained from degradome genes can predict the possibility of metastasis in patients with locally advanced cervical cancer.

Single Nucleotide Polymorphisms in Cellular Drug Transporters Are Associated with Intolerance to Antiretroviral Therapy in Brazilian HIV-1 Positive Individuals.

PubMed

Arruda, Mônica Barcellos; Campagnari, Francine; de Almeida, Tailah Bernardo; Couto-Fernandez, José Carlos; Tanuri, Amilcar; Cardoso, Cynthia Chester

2016-01-01

Adverse reactions are the main cause of treatment discontinuation among HIV+ individuals. Genes related to drug absorption, distribution, metabolism and excretion (ADME) influence drug bioavailability and treatment response. We have investigated the association between single nucleotide polymorphisms (SNPs) in 29 ADME genes and intolerance to therapy in a case-control study including 764 individuals. Results showed that 15 SNPs were associated with intolerance to nucleoside and 11 to non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and 8 to protease inhibitors (PIs) containing regimens under alpha = 0.05. After Bonferroni adjustment, two associations remained statistically significant. SNP rs2712816, at SLCO2B1 was associated to intolerance to NRTIs (ORGA/AA = 2.37; p = 0.0001), while rs4148396, at ABCC2, conferred risk of intolerance to PIs containing regimens (ORCT/TT = 2.64; p = 0.00009). Accordingly, haplotypes carrying rs2712816A and rs4148396T alleles were also associated to risk of intolerance to NRTIs and PIs, respectively. Our data reinforce the role of drug transporters in response to HIV therapy and may contribute to a future development of personalized therapies.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression.

PubMed

Leda, Ana Rachel; Hunter, James; Oliveira, Ursula Castro; Azevedo, Inacio Junqueira; Sucupira, Maria Cecilia Araripe; Diaz, Ricardo Sobhie

2018-04-19

The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies >20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies <14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+ T cell count decay. Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

PubMed

Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C; Mitsuya, Hiroaki; Sarafianos, Stefan G; Anderson, Karen S

2014-06-01

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. Copyright © 2014 Elsevier B.V. All rights reserved.

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) using pre-steady-state kinetics

PubMed Central

Muftuoglu, Yagmur; Sohl, Christal D.; Mislak, Andrea C.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Anderson, Karen S.

2014-01-01

The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. PMID:24632447

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, He, E-mail: herenrh@yahoo.com.cn; Zhao, Tiansuo; Wang, Xiuchao

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breastmore » cancer.« less

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

The origin and early evolution of nucleic acid polymerases

NASA Technical Reports Server (NTRS)

Lazcano, A.; Cappello, R.; Valverde, V.; Llaca, V.; Oro, J.

1992-01-01

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection.

PubMed

Giacobbi, Nicholas S; Sluis-Cremer, Nicolas

2017-07-01

Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. Copyright © 2017 American Society for Microbiology.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

2018-05-03

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X.

PubMed

Li, Fengwu; Bounkeua, Viengngeun; Pettersen, Kenneth; Vinetz, Joseph M

2016-02-24

Plasmodium invasion of the mosquito midgut is a population bottleneck in the parasite lifecycle. Interference with molecular mechanisms by which the ookinete invades the mosquito midgut is one potential approach to developing malaria transmission-blocking strategies. Plasmodium aspartic proteases are one such class of potential targets: plasmepsin IV (known to be present in the asexual stage food vacuole) was previously shown to be involved in Plasmodium gallinaceum infection of the mosquito midgut, and plasmepsins VII and plasmepsin X (not known to be present in the asexual stage food vacuole) are upregulated in Plasmodium falciparum mosquito stages. These (and other) parasite-derived enzymes that play essential roles during ookinete midgut invasion are prime candidates for transmission-blocking vaccines. Reverse transcriptase PCR (RT-PCR) was used to determine timing of P. falciparum plasmepsin VII (PfPM VII) and plasmepsin X (PfPM X) mRNA transcripts in parasite mosquito midgut stages. Protein expression was confirmed by western immunoblot and immunofluorescence assays (IFA) using anti-peptide monoclonal antibodies (mAbs) against immunogenic regions of PfPM VII and PfPM X. These antibodies were also used in standard membrane feeding assays (SMFA) to determine whether inhibition of these proteases would affect parasite transmission to mosquitoes. The Mann-Whitney U test was used to analyse mosquito transmission assay results. RT-PCR, western immunoblot and immunofluorescence assay confirmed expression of PfPM VII and PfPM X in mosquito stages. Whereas PfPM VII was expressed in zygotes and ookinetes, PfPM X was expressed in gametes, zygotes, and ookinetes. Antibodies against PfPM VII and PfPM X decreased P. falciparum invasion of the mosquito midgut when used at high concentrations, indicating that these proteases play a role in Plasmodium mosquito midgut invasion. Failure to generate genetic knockouts of these genes limited determination of the precise role of these proteases in parasite transmission but suggests that they are essential during the intraerythrocytic life cycle. PfPM VII and PfPM X are present in the mosquito-infective stages of P. falciparum. Standard membrane feeding assays demonstrate that antibodies against these proteins reduce the infectivity of P. falciparum for mosquitoes, suggesting their viability as transmission-blocking vaccine candidates. Further study of the role of these plasmepsins in P. falciparum biology is warranted.

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase

PubMed Central

Kopera, Huira C.; Moldovan, John B.; Morrish, Tammy A.; Moran, John V.

2011-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase. PMID:21940498

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

PubMed

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus.

PubMed

Wu, Xinghai; Chen, Chanfa; Xiao, Xizhi; Deng, Ming Jun

2016-11-01

A protocol for the reverse transcription-helicase-dependent amplification (RT-HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT-HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT-HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT-HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT-HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

Saquinavir-mediated inhibition of human immunodeficiency virus (HIV) infection in SCID mice implanted with human fetal thymus and liver tissue: an in vivo model for evaluating the effect of drug therapy on HIV infection in lymphoid tissues.

PubMed Central

Pettoello-Mantovani, M; Kollmann, T R; Raker, C; Kim, A; Yurasov, S; Tudor, R; Wiltshire, H; Goldstein, H

1997-01-01

Treatment with protease inhibitors alone or in combination with inhibitors of reverse transcriptase potently suppresses levels of human immunodeficiency virus (HIV) RNA in plasma and thereby may significantly delay the progression of HIV-mediated disease. To investigate the effect of treatment with the protease inhibitor saquinavir on HIV replication in the lymphoid tissues, we used a SCID-hu mouse model that we developed, in which human thymic and liver tissues (hu-thy/liv) were implanted under both kidney capsules in SCID mice (thy/liv-SCID-hu mice). These mice are populated in the periphery with large numbers of human T cells and develop disseminated HIV infection after intraimplant injection. thy/liv-SCID-hu mice with established HIV infection that were treated for 1 month with saquinavir had a significantly lower viral load present in the implanted hu-thy/liv and mouse spleen than did the untreated HIV-infected thy/liv-SCID-hu mice. To examine the capacity of acute treatment with saquinavir to prevent HIV infection, some thy/liv-SCID-hu mice were inoculated with HIV and then immediately started on saquinavir. Although treated mice had markedly lower viral loads in the thy/liv implants and spleens, HIV infection was not completely prevented. Thus, the effect of antiviral therapy on HIV infection in the major site of HIV replication, the lymphoid tissues, can be readily evaluated in our thy/liv-SCID-hu mice. These mice should prove to be a useful model for determining the in vivo effectiveness of different therapeutic interventions on acute and chronic HIV infection. PMID:9303378

Nucleoside Analog 1592U89 and Human Immunodeficiency Virus Protease Inhibitor 141W94 Are Synergistic In Vitro

PubMed Central

Drusano, G. L.; D’Argenio, D. Z.; Symonds, W.; Bilello, P. A.; McDowell, J.; Sadler, B.; Bye, A.; Bilello, J. A.

1998-01-01

The use of combinations of anti-human immunodeficiency virus (anti-HIV) agents targeted to different molecular targets will most likely result in increased viral suppression and may also delay or prevent the emergence of resistant HIV strains. The purpose of the present study was to develop information on the in vitro anti-HIV activities of combinations of the reverse transcriptase inhibitor 1592U89 and the protease inhibitor 141W94 to help guide the choice of dosages in clinical trials. Triplicate in vitro dose-response matrices were prepared with MT-2 cells infected with HIV type 1 (HIV-1) strain IIIB. In order to account for the effects of protein binding, tissue culture medium with 10% fetal bovine serum was supplemented with the human serum proteins α1 acid glycoprotein (1 mg/ml) and albumin (40 mg/ml). The three-dimensional drug interaction surface for 1592U89 and 141W94 was constructed with the program MacSynergy II. As analyzed relative to a Bliss Independence null reference model, this combination was synergistic, with volumes of synergy exceeding 100 (99% confidence). Analysis of the data set with a fully parametric form of an equation for the quantitation of drug interaction developed by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331–385, 1995) resulted in an interaction term statistically significantly greater than 0.0, indicating true synergy. Both methods concur that this combination is significantly synergistic. These data, with favorable findings from phase I/II trials for each drug alone, suggest that the combination of 1592U89 plus 141W94 should be further evaluated in clinical trials. PMID:9736527

Highly active antiretroviral therapy including protease inhibitors does not confer a unique CD4 cell benefit. The AVANTI and INCAS Study Groups.

PubMed

2000-07-07

To determine if triple combination therapy, particularly including HIV protease inhibitors (PI), confers an unique immunological benefit that is independent of reductions of plasma viral load (pVL). The correlation between changes from baseline in CD4 cell count and pVL was examined at all time points up to 52 weeks in three randomized clinical trials (AVANTI-2, AVANTI-3 and INCAS) that compared dual nucleoside therapy with triple combination therapy. Individual pVL and CD4 cell counts changes from baseline were entered into multivariate linear regression models for patients receiving double therapy and for those receiving triple therapy including a PI and/or a non-nucleoside reverse transcriptase inhibitor (NNRTI), and the null hypothesis was tested. After 52 weeks of therapy, the relationship between changes from baseline CD4 cell count and pVL was independent of whether patients were assigned double or triple therapy (P = 0.23 and 0.69 for intercept and slope, respectively), or whether patients were assigned triple therapy including a PI or triple therapy including an NNRTI (P = 0.92 and 0.95, respectively). Less than 5% of patients ever had 'discordant' increases in both CD4 cell count and pVL compared with baseline, and this proportion was unrelated to the class of therapy used. 'Discordant' decreases from baseline in both parameters were observed in up to 35% of individuals. The correlation between pVL and CD4 cell count changes from baseline improved over time on therapy, regardless of the therapeutic regimen involved. The data provide no evidence for a CD4 cell count benefit of highly active antiretroviral therapy (HAART) unique to triple therapy or PI-containing regimens.

Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs.

PubMed

Ding, Feng; Miao, Xi-Li; Li, Yan-Xia; Dai, Jin-Fen; Yu, Hong-Gang

2016-01-01

The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. This research aimed to determine the clinical and virological features of the rare pattern. A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. The amino acid variability within major hydrophilic region, especially the "a" determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the "a" determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Trends of drug-resistance-associated mutations in the reverse transcriptase gene of HIV type 1 isolates from North India.

PubMed

Azam, Mohd; Malik, Abida; Rizvi, Meher; Rai, Arvind

2014-04-01

A major cause of failure of antiretroviral therapy (ART) is the presence of drug-resistance-associated mutations in the polymerase gene of HIV-1. The paucity of data regarding potential drug resistance to reverse transcriptase inhibitors (RTIs) prompted us to carry out this study. This information will shed light on the extent of drug resistance already present in HIV strains and will give future directions in patient treatment and in drug design. Drug resistance genotyping of a partial reverse transcriptase gene was done in 103 HIV-1-infected patients, including the ART-naive and ART-experienced population. The drug resistance pattern was analyzed using the Stanford HIV-DR database, the IAS-USA mutation list and the REGA algorithm-v8.0. Subtyping was done using the REGA HIV-1 subtyping tool-v2.01. The majority of our sequences (96 %) were found to be subtype C, and four (3.8 %) were subtype A1. Significant prevalence of DR mutations (28 %) was observed in the RT gene. Major amino acid substitutions were seen at positions 41, 90, 98, 103, 106, 108, 138, 181, 184, 190, 215, and 219, which confer high/intermediate levels of resistance to most RTIs, independently or together. Our results show that there is an urgent need to tailor ART drug regimens to the individual to achieve optimum therapeutic outcome in North India.

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

PubMed

Ahmad, Fahim; Patrick, Shruti; Sheikh, Touseef; Sharma, Vikas; Pathak, Pankaj; Malgulwar, Prit Benny; Kumar, Anupam; Joshi, Shanker Datt; Sarkar, Chitra; Sen, Ellora

2017-12-01

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with in vitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma. © 2017 International Society for Neurochemistry.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

PubMed

Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

2016-07-01

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat. Copyright © 2016 Nowicka-Sans et al.

Deregulation of the telomerase reverse transcriptase (TERT) gene by chromosomal translocations in B-cell malignancies.

PubMed

Nagel, Inga; Szczepanowski, Monika; Martín-Subero, José I; Harder, Lana; Akasaka, Takashi; Ammerpohl, Ole; Callet-Bauchu, Evelyne; Gascoyne, Randy D; Gesk, Stefan; Horsman, Doug; Klapper, Wolfram; Majid, Aneela; Martinez-Climent, José A; Stilgenbauer, Stephan; Tönnies, Holger; Dyer, Martin J S; Siebert, Reiner

2010-08-26

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.

Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

PubMed

Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2016-06-01

Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

Hepatotoxicity of nucleoside reverse transcriptase inhibitors.

PubMed

Montessori, Valentina; Harris, Marianne; Montaner, Julio S G

2003-05-01

Hepatotoxicity is an adverse effect of all available classes of antiretrovirals, including nucleoside reverse transcriptase inhibitors (NRTI). A syndrome of hepatic steatosis and lactic acidosis has been recognized as a rare, potentially fatal complication since the advent of NRTI monotherapy in the early 1990s. Today, NRTI remain the backbone of antiretroviral combination regimens, and, with the success of current treatment strategies, exposure to two or more of these agents may occur over a number of years. Hepatic steatosis and lactic acidosis are accordingly being observed more frequently, along with a more recently recognized syndrome of chronic hyperlactatemia. These as well as other adverse effects of NRTI are mediated by inhibition of human DNA polymerase gamma, resulting in mitochondrial dysfunction in the liver and other tissues. Early recognition and intervention are essential to avert serious outcomes.

Herpes Simplex Virus Type 2 Suppressive Therapy with Acyclovir or Valacyclovir Does Not Select for Specific HIV-1 Resistance in HIV-1/HSV-2 Dually Infected Persons

PubMed Central

Lingappa, Jairam; Beck, Ingrid; Frenkel, Lisa M.; Pepper, Gregory; Celum, Connie; Wald, Anna; Fife, Kenneth H.; Were, Edwin; Mugo, Nelly; Sanchez, Jorge; Essex, Myron; Makhema, Joseph; Kiarie, James; Farquhar, Carey; Corey, Lawrence

2011-01-01

Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1–infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%–2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance. PMID:21148504

Perinatal exposure of patas monkeys to antiretroviral nucleoside reverse-transcriptase inhibitors induces genotoxicity persistent for up to 3 years of age.

PubMed

Olivero, Ofelia A; Torres, Lorangelly Rivera; Gorjifard, Sayeh; Momot, Dariya; Marrogi, Eryney; Divi, Rao L; Liu, Yongmin; Woodward, Ruth A; Sowers, Marsha J; Poirier, Miriam C

2013-07-15

Erythrocebus patas (patas) monkeys were used to model antiretroviral (ARV) drug in human immunodeficiency virus type 1-infected pregnant women. Pregnant patas dams were given human-equivalent doses of ARVs daily during 50% of gestation. Mesenchymal cells, cultured from bone marrow of patas offspring obtained at birth and at 1 and 3 years of age, were examined for genotoxicity, including centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes. Compared with controls, statistically significant increases (P < .05) in centrosomal amplification, micronuclei, and micronuclei containing whole chromosomes were found in mesenchymal cells from most groups of offspring at the 3 time points. Transplacental nucleoside reverse-transcriptase inhibitor exposures induced fetal genotoxicity that was persistent for 3 years.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

HIV/AIDS Medicines

MedlinePlus

... few years. But today, there are many effective medicines to fight the infection, and people with HIV ... healthier lives. There are five major types of medicines: Reverse transcriptase (RT) inhibitors - interfere with a critical ...

Expression of TGF-beta1, osteonectin, and BMP-4 in mandibular distraction osteogenesis with compression stimulation: reverse transcriptase-polymerase chain reaction study and biomechanical test.

PubMed

Kim, Uk-Kyu; Park, Seong-Jin; Seong, Wook-Jin; Heo, Jun; Hwang, Dae-Seok; Kim, Yong-Deok; Shin, Sang-Hun; Kim, Gyoo-Cheon

2010-09-01

This study compared the levels of transforming growth factor-beta1 (TGF-beta1), osteonectin, and bone morphogenetic protein-4 (BMP-4) expression in regenerated bone in a rabbit mandible that had undergone conventional distraction osteogenesis (DO) with those in regenerated bone from a modified DO technique with compression stimulation. A total of 42 rabbits were used in this reverse transcriptase-polymerase chain reaction study. In the control group, distraction was performed at 1 mm/day for 8 days. In the experimental group, overdistraction was performed for 10 days, followed by a 3-day latency period and 2 days of compression to achieve the same amount of DO. Three rabbits per subgroup were killed at 0, 5, 13, 20, 27, 34, and 41 days after the initial osteotomy. The levels of TGF-beta1, osteonectin, and BMP-4 in the bone regenerates were measured by reverse transcriptase-polymerase chain reaction. A biomechanical microhardness test was also performed in 8 rabbits as a separate experiment. Reverse transcriptase-polymerase chain reaction revealed a greater level of TGF-beta1 in the experimental group immediately after applying the compression force that continued for 2 weeks. The level then decreased to that of the control group at 3 weeks. The greater level of osteonectin in the experimental group after compression than that in the control group continued for 3 weeks. In the experimental group, the level of BMP-4 increased immediately after compression. However, the level in the control group decreased. The microhardness ratio of distracted bone to normal bone on the cortex was statistically different at 0.47 in the control group and 0.80 in the experimental group (P = .049) at 55 days after osteotomy. The effectiveness of the new DO technique with compression stimulation was confirmed by the gene expression study and the biomechanical test findings. Copyright 2010 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Development and evaluation of a simple and effective RT-qPCR inhibitory assay for detection of the efficacy of compounds towards HIV reverse transcriptase.

PubMed

Marino-Merlo, Francesca; Frezza, Caterina; Papaianni, Emanuela; Valletta, Elena; Mastino, Antonio; Macchi, Beatrice

2017-11-01

Assessing the actual efficacy of compounds to directly inhibit HIV reverse transcriptase (RT) activity is a main goal in preclinical antiretroviral studies. Our previous studies demonstrated that the effects of inhibitor compounds towards HIV-RT could be efficiently assessed through a simple cell-free assay based on conventional reverse transcription PCR. In the present study, we describe a modified variant of our assay, termed RT real-time quantitative PCR inhibitory assay (RT-qPCR-IA), in which the ability of compounds to restrict the complementary DNA (cDNA) generation by HIV-RT using a specific RNA template is performed by the real-time technique, in order to improve both accuracy and sensitivity of the method. As specific RNA template, RNA extracted from stable transfectants ectopically expressing the herpes simplex virus 1 glycoprotein D gene was utilized. HIV-RT, of both commercial or house-made viral lysate origin, was employed for the assay. To assess the reliability of RT-qPCR-IA, we performed a comparative, quantitative analysis of the dose-dependent effect exerted by known nucleotide and non-nucleotide reverse-transcriptase inhibitors, using the SYBR Green dye chemistry as detection system. The results obtained with RT-qPCR-IA were compared to that obtained using a one-step PicoGreen technology-based commercial kit. The outcome of our study indicates that the development of the novel RT-qPCR-IA will provide rapid and accurate evaluation of the inhibitory efficacy of compounds towards HIV-RT activity. This evaluation could be very useful for large-scale screening of potential new anti-HIV drugs.

Synthesis and biological evaluation of 2-thioxopyrimidin-4(1H)-one derivatives as potential non-nucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Khalifa, Nagy M; Al-Omar, Mohamed A

2014-11-12

A series of new 5-allyl-6-benzylpyrimidin-4(3H)-ones bearing different substituents at the C-2 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against human immunodeficiency virus type 1 (HIV-1) in the human T-lymphotropic type (MT-4 cell cultures). The majority of the title compounds showed moderate to good activities against HIV-1. Amongst them, 5-allyl-6-benzyl-2-(3-hydroxypropylthio)pyrimidin-4(3H)-one analogue 11c exhibited the most potent anti-HIV-1 activity (IC50 0.32 µM). The biological testing results clearly indicated that the substitution at C-2 position of the pyrimidine ring could increase the anti-HIV-1 reverse transcriptase (RT) activity.

Synthesis and Biological Evaluation of 2-Thioxopyrimidin-4(1H)-one Derivatives as Potential Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors

PubMed Central

Khalifa, Nagy M.; Al-Omar, Mohamed A.

2014-01-01

A series of new 5-allyl-6-benzylpyrimidin-4(3H)-ones bearing different substituents at the C-2 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against human immunodeficiency virus type 1 (HIV-1) in the human T-lymphotropic type (MT-4 cell cultures). The majority of the title compounds showed moderate to good activities against HIV-1. Amongst them, 5-allyl-6-benzyl-2-(3-hydroxypropylthio)pyrimidin-4(3H)-one analogue 11c exhibited the most potent anti-HIV-1 activity (IC50 0.32 µM). The biological testing results clearly indicated that the substitution at C-2 position of the pyrimidine ring could increase the anti-HIV-1 reverse transcriptase (RT) activity. PMID:25397597

Complete inactivation of HIV-1 using photo-labeled non-nucleoside reverse transcriptase inhibitors.

PubMed

Rios, Adan; Quesada, Jorge; Anderson, Dallas; Goldstein, Allan; Fossum, Theresa; Colby-Germinario, Susan; Wainberg, Mark A

2011-01-01

We demonstrate that a photo-labeled derivative of the non-nucleoside reverse transcriptase inhibitor (NNRTI) dapivirine termed DAPY, when used together with exposure to ultraviolet light, was able to completely and irreversibly inactivate both HIV-1 RT activity as well as infectiousness in each of a T cell line and peripheral blood mononuclear cells. Control experiments using various concentrations of DAPY revealed that a combination of exposure to ultraviolet light together with use of the specific, high affinity photo-labeled compound was necessary for complete inactivation to occur. This method of HIV RT inactivation may have applicability toward preservation of an intact viral structure and warrants further investigation in regard to the potential of this approach to elicit a durable, broad protective immune response. Copyright © 2010 Elsevier B.V. All rights reserved.

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

PubMed

Monforte, Anna Maria; De Luca, Laura; Buemi, Maria Rosa; Agharbaoui, Fatima E; Pannecouque, Christophe; Ferro, Stefania

2018-02-01

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N 1 -aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N 1 -aryl-2-arylthioacetamido-benzimidazoles and N 1 -aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

PubMed

Pontikis, R; Dollé, V; Guillaumel, J; Dechaux, E; Note, R; Nguyen, C H; Legraverend, M; Bisagni, E; Aubertin, A M; Grierson, D S; Monneret, C

2000-05-18

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

PubMed

AboElkhair, M; Iwamoto, T; Clark, K F; McKenna, P; Siah, A; Greenwood, S J; Berthe, F C J; Casey, J W; Cepica, A

2012-01-01

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences. Copyright © 2011 Elsevier Inc. All rights reserved.

Vascular endothelial growth factor and platelet-derived growth factor are potential angiogenic and metastatic factors in human breast cancer.

PubMed

Anan, K; Morisaki, T; Katano, M; Ikubo, A; Kitsuki, H; Uchiyama, A; Kuroki, S; Tanaka, M; Torisu, M

1996-03-01

Angiogenesis is a prerequisite for tumor growth and metastasis. Tumor angiogenesis may be mediated by several angiogenic factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha, and basic fibroblast growth factor. Differential mRNA expressions of VEGF, PDGF (A chain), transforming growth factor-alpha and basic fibroblast growth factor in 32 primary invasive breast tumors were examined by reverse transcriptase-polymerase chain reaction. We analyzed relationships between mRNA expressions of these angiogenic factors and the degree of angiogenesis, tumor size, and metastasis. Quantification of angiogenesis was achieved by the immunohistochemical staining of endothelial cells with antibody to CD31. VEGF and PDGF-A mRNAs were expressed more frequently in breast tumors than in nontumor breast tissues, whereas no difference was found in expression frequency of either transforming growth factor-alpha or basic fibroblast growth factor mRNA. Vascular counts in tumors correlated with each expression frequency of VEGF and PDGF-A mRNA. PDGF-A mRNA was expressed more frequently in tumors with lymph node metastasis than in those without metastasis. Expression of VEGF and PDGF mRNAs detected by reverse transcriptase-polymerase chain reaction in breast tumors correlates with tumor-related characteristics of angiogenesis and metastatic potential. Analysis of these mRNAs by reverse transcriptase-polymerase chain reaction may be useful for assessing the biologic behavior of a breast tumor before surgical treatment.

HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

PubMed

Poppe, Lisa K; Chunda-Liyoka, Catherine; Kwon, Eun H; Gondwe, Clement; West, John T; Kankasa, Chipepo; Ndongmo, Clement B; Wood, Charles

2017-08-24

The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

In Vitro Resistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine

PubMed Central

Schader, Susan M.; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P.

2012-01-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries. PMID:22123692

In vitro resistance profile of the candidate HIV-1 microbicide drug dapivirine.

PubMed

Schader, Susan M; Oliveira, Maureen; Ibanescu, Ruxandra-Ilinca; Moisi, Daniela; Colby-Germinario, Susan P; Wainberg, Mark A

2012-02-01

Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.

Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

USDA-ARS?s Scientific Manuscript database

Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

USDA-ARS?s Scientific Manuscript database

Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

Reverse transcription of phage RNA and its fragment directed by synthetic heteropolymeric primers

PubMed Central

Frolova, L. Yu.; Metelyev, V. G.; Ratmanova, K. I.; Smirnov, V. D.; Shabarova, Z. A.; Prokofyev, M. A.; Berzin, V. M.; Jansone, I. V.; Gren, E. J.; Kisselev, L. L.

1977-01-01

DNA synthesis catalysed by RNA-directed DNA-polymerase (reverse transcriptase) was found to proceed on the RNA template of an MS2 phage in the presence of heteropolymeric synthetic octa- and nonadeoxyribonucleotide primers complementary to the intercistronic region (coat protein binding site) and the region of the coat protein cistron, respectively. The product of synthesis consists of discrete DNA fractions of different length, including transcripts longer than 1,000 nucleotides. The coat protein inhibits DNA synthesis if it is initiated at its binding site, but has no effect on DNA synthesis initiated at the coat protein cistron. It has been suggested that, in this system, the initiation of DNA synthesis by synthetic primers is topographically specific. The MS2 coat protein binding site (an RNA fragment of 59 nucleotides) serves as a template for polydeoxyribonucleotide synthesis in the presence of octanucleotide primer and reverse transcriptase. The product of synthesis is homogenous and its length corresponds to the length of the template. The effective and complete copying of the fragment having a distinct secondary structure proves that the secondary structure does not interfere, in principle, with RNA being a template in the system of reverse transcription. PMID:71713

Global asymptotic stability for HIV-1 dynamics with two distributed delays.

PubMed

Wang, Jinliang; Huang, Gang; Takeuchi, Yasuhiro

2012-09-01

Based on the drugs treatment to control HIV-1 infection and viral replication, we express the intracellular eclipse phase of virions in host cell as distributed delays because of pharmacological actions. In present paper, we investigate a class of HIV-1 infection dynamical model with two distributed delays. One of them describes the period between the time that HIV virion enters (infects) target cell and the time that the infected cell starts to produce new viral particles. The other describes the time for the virion maturation process. They are both allowed to tend to be infinite because of drugs resistent strains. By the Lyapunov direct method of and utilizing the technology of constructing Lyapunov functionals, we identify the basic reproduction number R(0) as a threshold quantity for the stability of equilibria. More precisely, if R(0) ≤ 1, the infection-free equilibrium is globally asymptotically stable; on the contrary, if R(0) > 1, then an infected equilibrium appears which is globally asymptotically stable. The dynamical results indicate that time delays have effect on the global stability of two equilibria through threshold value R(0), which is a decreasing function of delays. The biological meanings imply that any drugs that can prolong the time of viral reproduction through slowing down the reverse transcription of HIV in host and virus maturation process may also help control the HIV-1 infection and virus loads. Another way to increase the efficacy of the protease inhibitor and the reverse transcriptase inhibitor (i.e. increasing n(p) and n(rt)) is also desirable treatment strategies.

Genome-Wide Profiling of RNA–Protein Interactions Using CLIP-Seq

PubMed Central

Stork, Cheryl; Zheng, Sika

2017-01-01

UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein–RNA interactions in tissues and cells. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein–epitope tag is then used to immunoprecipitate the protein–RNA complexes. After stringent washing and gel separation the RBP–RNA complex is excised. The RBP is protease digested to allow purification of the bound RNA. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. Here, we describe the detailed procedure of iCLIP. PMID:26965263

Risk factors for pre-term birth in a Canadian cohort of HIV-positive women: role of ritonavir boosting?

PubMed Central

Kakkar, Fatima; Boucoiran, Isabelle; Lamarre, Valerie; Ducruet, Thierry; Amre, Devendra; Soudeyns, Hugo; Lapointe, Normand; Boucher, Marc

2015-01-01

Background The risk of pre-term birth (PTB) associated with the use of protease inhibitors (PIs) during pregnancy remains a subject of debate. Recent data suggest that ritonavir boosting of PIs may play a specific role in the initiation of PTB, through an effect on the maternal–fetal adrenal axis. The primary objective of this study is to compare the risk of PTB among women treated with boosted PI versus non-boosted PIs during pregnancy. Methods Between 1988 and 2011, 705 HIV-positive women were enrolled into the Centre Maternel et Infantile sur le SIDA mother–infant cohort at Centre Hospitalier Universitaire Sainte-Justine in Montreal, Canada. Inclusion criteria for the study were: 1) attendance at a minimum of two antenatal obstetric visits and 2) singleton live birth, at 24 weeks gestational or older. The association between PTB (defined as delivery at <37 weeks gestational age), antiretroviral drug exposure and maternal risk factors was assessed retrospectively using logistic regression. Results A total of 525 mother–infant pairs were included in the analysis. Among them, PI-based combination anti-retroviral therapy was used in 37.4%, boosted PI based in 24.4%, non-nucleoside reverse transcriptase inhibitor (NNRTI) or nucleoside reverse transcriptase inhibitor based in 28.1%, and no treatment was given in 10.0% of cases. Overall, 13.5% of women experienced PTB. Among women treated with antiretroviral therapy, the risk of PTB was significantly higher among women who received boosted versus non-boosted PI (OR 2.01, 95% CI 1.02–3.97). This remained significant after adjusting for maternal age, delivery CD4 count, hepatitis C co-infection, history of previous PTB, and parity (aOR 2.17, 95% CI 1.05–4.51). There was no increased risk of PTB with the use of unboosted PIs as compared to NNRTI- or NRTI-based regimens. Conclusion While previous studies on the association between PTB and PI use have generally considered all PIs the same, our results would indicate a possible role of ritonavir boosting as a risk factor for PTB. Further work is needed to understand the pathophysiologic mechanisms involved, and to identify the safest ARV regimens to be used in pregnancy. PMID:26051165

[GESIDA/National AIDS Plan: Consensus document on antiretroviral therapy in adults infected by the human immunodeficiency virus (Updated January 2015)].

PubMed

2015-10-01

This consensus document is an update of combined antiretroviral therapy (cART) guidelines and recommendations for HIV-1 infected adult patients. To formulate these recommendations, a panel composed of members of the AIDS Study Group and the AIDS National Plan (GeSIDA/Plan Nacional sobre el Sida) reviewed the efficacy and safety advances in clinical trials, and cohort and pharmacokinetic studies published in medical journals (PubMed and Embase) or presented in medical scientific meetings. The strength of the recommendations, and the evidence that supports them, are based on modified criteria of the Infectious Diseases Society of America. In this update, cART is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The strength and level of the recommendation depends on the CD4+T-lymphocyte count, the presence of opportunistic diseases or comorbid conditions, age, and prevention of transmission of HIV. The objective of cART is to achieve an undetectable plasma viral load. Initial cART should always comprise a combination of 3 drugs, including 2 nucleoside reverse transcriptase inhibitors, and a third drug from a different family. Three out of the ten recommended regimes are regarded as preferential (all of them with an integrase inhibitor as the third drug), and the other seven (based on a non-nucleoside reverse transcriptase inhibitor, a ritonavir-boosted protease inhibitor, or an integrase inhibitor) as alternatives. This update presents the causes and criteria for switching cART in patients with undetectable plasma viral load, and in cases of virological failure where rescue cART should comprise 3 (or at least 2) drugs that are fully active against the virus. An update is also provided for the specific criteria for cART in special situations (acute infection, HIV-2 infection, and pregnancy) and with comorbid conditions (tuberculosis or other opportunistic infections, kidney disease, liver disease, and cancer). These new guidelines update previous recommendations related to cART (when to begin and what drugs should be used), how to monitor and what to do in case of viral failure or drug adverse reactions. cART specific criteria in comorbid patients and special situations are equally updated. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Higher rates of triple-class virological failure in perinatally HIV-infected teenagers compared with heterosexually infected young adults in Europe.

PubMed

Judd, A; Lodwick, R; Noguera-Julian, A; Gibb, D M; Butler, K; Costagliola, D; Sabin, C; van Sighem, A; Ledergerber, B; Torti, C; Mocroft, A; Podzamczer, D; Dorrucci, M; De Wit, S; Obel, N; Dabis, F; Cozzi-Lepri, A; García, F; Brockmeyer, N H; Warszawski, J; Gonzalez-Tome, M I; Mussini, C; Touloumi, G; Zangerle, R; Ghosn, J; Castagna, A; Fätkenheuer, G; Stephan, C; Meyer, L; Campbell, M A; Chene, G; Phillips, A

2017-03-01

The aim of the study was to determine the time to, and risk factors for, triple-class virological failure (TCVF) across age groups for children and adolescents with perinatally acquired HIV infection and older adolescents and adults with heterosexually acquired HIV infection. We analysed individual patient data from cohorts in the Collaboration of Observational HIV Epidemiological Research Europe (COHERE). A total of 5972 participants starting antiretroviral therapy (ART) from 1998, aged < 20 years at the start of ART for those with perinatal infection and 15-29 years for those with heterosexual infection, with ART containing at least two nucleoside reverse transcriptase inhibitors (NRTIs) and a nonnucleoside reverse transcriptase inhibitor (NNRTI) or a boosted protease inhibitor (bPI), were followed from ART initiation until the most recent viral load (VL) measurement. Virological failure of a drug was defined as VL > 500 HIV-1 RNA copies/mL despite ≥ 4 months of use. TCVF was defined as cumulative failure of two NRTIs, an NNRTI and a bPI. The median number of weeks between diagnosis and the start of ART was higher in participants with perinatal HIV infection compared with participants with heterosexually acquired HIV infection overall [17 (interquartile range (IQR) 4-111) vs. 8 (IQR 2-38) weeks, respectively], and highest in perinatally infected participants aged 10-14 years [49 (IQR 9-267) weeks]. The cumulative proportion with TCVF 5 years after starting ART was 9.6% [95% confidence interval (CI) 7.0-12.3%] in participants with perinatally acquired infection and 4.7% (95% CI 3.9-5.5%) in participants with heterosexually acquired infection, and highest in perinatally infected participants aged 10-14 years when starting ART (27.7%; 95% CI 13.2-42.1%). Across all participants, significant predictors of TCVF were those with perinatal HIV aged 10-14 years, African origin, pre-ART AIDS, NNRTI-based initial regimens, higher pre-ART viral load and lower pre-ART CD4. The results suggest a beneficial effect of starting ART before adolescence, and starting young people on boosted PIs, to maximize treatment response during this transitional stage of development. © 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

First Line Treatment Response in Patients with Transmitted HIV Drug Resistance and Well Defined Time Point of HIV Infection: Updated Results from the German HIV-1 Seroconverter Study

PubMed Central

zu Knyphausen, Fabia; Scheufele, Ramona; Kücherer, Claudia; Jansen, Klaus; Somogyi, Sybille; Dupke, Stephan; Jessen, Heiko; Schürmann, Dirk; Hamouda, Osamah; Meixenberger, Karolin; Bartmeyer, Barbara

2014-01-01

Background Transmission of drug-resistant HIV-1 (TDR) can impair the virologic response to antiretroviral combination therapy. Aim of the study was to assess the impact of TDR on treatment success of resistance test-guided first-line therapy in the German HIV-1 Seroconverter Cohort for patients infected with HIV between 1996 and 2010. An update of the prevalence of TDR and trend over time was performed. Methods Data of 1,667 HIV-infected individuals who seroconverted between 1996 and 2010 were analysed. The WHO drug resistance mutations list was used to identify resistance-associated HIV mutations in drug-naïve patients for epidemiological analysis. For treatment success analysis the Stanford algorithm was used to classify a subset of 323 drug-naïve genotyped patients who received a first-line cART into three resistance groups: patients without TDR, patients with TDR and fully active cART and patients with TDR and non-fully active cART. The frequency of virologic failure 5 to 12 months after treatment initiation was determined. Results Prevalence of TDR was stable at a high mean level of 11.9% (198/1,667) in the HIV-1 Seroconverter Cohort without significant trend over time. Nucleotide reverse transcriptase inhibitor resistance was predominant (6.0%) and decreased significantly over time (OR = 0.92, CI = 0.87–0.98, p = 0.01). Non-nucleoside reverse transcriptase inhibitor (2.4%; OR = 1.00, CI = 0.92–1.09, p = 0.96) and protease inhibitor resistance (2.0%; OR = 0.94, CI = 0.861.03, p = 0.17) remained stable. Virologic failure was observed in 6.5% of patients with TDR receiving fully active cART, 5,6% of patients with TDR receiving non-fully active cART and 3.2% of patients without TDR. The difference between the three groups was not significant (p = 0.41). Conclusion Overall prevalence of TDR remained stable at a rather high level. No significant differences in the frequency of virologic failure were identified during first-line cART between patients with TDR and fully-active cART, patients with TDR and non-fully active cART and patients without TDR. PMID:24788613

Bacterial Group II Introns: Identification and Mobility Assay.

PubMed

Toro, Nicolás; Molina-Sánchez, María Dolores; Nisa-Martínez, Rafael; Martínez-Abarca, Francisco; García-Rodríguez, Fernando Manuel

2016-01-01

Group II introns are large catalytic RNAs and mobile retroelements that encode a reverse transcriptase. Here, we provide methods for their identification in bacterial genomes and further analysis of their splicing and mobility capacities.

The discovery of a class of novel HIV-1 maturation inhibitors and their potential in the therapy of HIV.

PubMed

Yu, Donglei; Wild, Carl T; Martin, David E; Morris-Natschke, Susan L; Chen, Chin-Ho; Allaway, Graham P; Lee, Kuo-Hsiung

2005-06-01

Although HIV infection is now primarily treated with reverse transcriptase and protease inhibitors, HIV therapy must look toward new drugs with novel mechanism(s) of action to both improve efficacy and address the growing problem of drug resistance. Using natural products as a source of biologically active compounds, our drug discovery program has successfully optimised the natural product betulinic acid to the first-in-class maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB). DSB's unique viral target has been identified as a late step in Gag processing. Specifically, it inhibits the cleavage of the capsid precursor, CA-SP1, resulting in a block to the processing of mature capsid protein leading to a defect in viral core condensation. DSB represents a unique class of anti-HIV compounds that inhibit virus maturation and provide additional opportunities for anti-HIV therapy. In this review, the discovery of DSB and its mode of action are summarised. Anti-AIDS Agents part 64. For part 63 in the series, see YU D, LEE KH: Recent progress and prospects on plant-derived anti-HIV agents and analogs. In: Medicinal Chemistry of Bioactive Natural Products. XT Liang, WS Fang (Eds), Wiley, New York, USA (2005) (In Press).

Targets for inhibition of HIV replication: entry, enzyme action, release and maturation.

PubMed

Sierra-Aragón, Saleta; Walter, Hauke

2012-01-01

Inhibition of HIV replication initially targeted viral enzymes, which are exclusively expressed by the virus and not present in the human cell. The development of reverse transcriptase (RT) inhibitors started with the discovery of antiretroviral activity of the nucleoside analog zidovudine in March 1987. Currently, six major classes of antiretroviral drugs are used for the treatment of HIV-infected patients: the RT inhibitors, nucleoside inhibitors and nonnucleoside inhibitors, the protease inhibitors, the integrase inhibitor raltegravir, the fusion inhibitor enfuvirtide (T-20), and the chemokine receptor 5 antagonist maraviroc. A seventh class, the maturation inhibitors, has not yet been approved as their effectiveness is impaired by HIV-1 polymorphisms naturally occurring in 30-40% of HIV-1 therapy-naive isolates. The use of antiretroviral combination therapy has proven to be effective in delaying progression to AIDS and to reconstitute the immune system of HIV-infected individuals. During the last 5 years, the introduction of the newest antiretrovirals has increased treatment efficacy tremendously. However, the development and accumulation of resistance to all antiretroviral drug classes are still a major problem. Additional targets will have to be defined to achieve the ultimate goal: the eradication of the virus from the infected human body. Copyright © 2012 S. Karger AG, Basel.

Antiretroviral Drug Susceptibility Among HIV-Infected Adults Failing Antiretroviral Therapy in Rakai, Uganda

PubMed Central

Laeyendecker, Oliver; Nakigozi, Gertrude; Gallant, Joel E.; Huang, Wei; Hudelson, Sarah E.; Quinn, Thomas C.; Newell, Kevin; Serwadda, David; Gray, Ronald H.; Wawer, Maria J.; Eshleman, Susan H.

2012-01-01

Abstract We analyzed antiretroviral drug susceptibility in HIV-infected adults failing first- and second-line antiretroviral treatment (ART) in Rakai, Uganda. Samples obtained from participants at baseline (pretreatment) and at the time of failure on first-line ART and second-line ART were analyzed using genotypic and phenotypic assays for antiretroviral drug resistance. Test results were obtained from 73 samples from 38 individuals (31 baseline samples, 36 first-line failure samples, and six second-line failure samples). Four (13%) of the 31 baseline samples had mutations associated with resistance to nucleoside or nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively). Among the 36 first-line failure samples, 31 (86%) had NNRTI resistance mutations and 29 (81%) had lamivudine resistance mutations; only eight (22%) had other NRTI resistance mutations. None of the six individuals failing a second-line protease inhibitor (PI)-based regimen had PI resistance mutations. Six (16%) of the participants had discordant genotypic and phenotypic test results. Genotypic resistance to drugs included in first-line ART regimens was detected prior to treatment and among participants failing first-line ART. PI resistance was not detected in individuals failing second-line ART. Surveillance for transmitted and acquired drug resistance remains a priority for scale-up of ART. PMID:22443282

Sensitivity Changes over the Course of Infection Increases the Likelihood of Resistance Against Fusion but Not CCR5 Receptor Blockers

PubMed Central

Chatziandreou, Nikolaos; Arauz, Ana Belen; Freitas, Ines; Nyein, Phyu Hninn; Fenton, Gregory; Mehta, Shruti H.; Kirk, Gregory D.

2012-01-01

Abstract As HIV-1 evolves over the course of infection, resistance against antiretrovirals may arise in the absence of drug pressure, especially against receptor and fusion blockers because of the extensive changes observed in the envelope glycoprotein. Here we show that viruses from the chronic phase of disease are significantly less sensitive to CCR5 receptor and fusion blockers compared to early infection variants. Differences in susceptibility to CCR5 antagonists were observed in spite of no demonstrable CXCR4 receptor utilization. No significant sensitivity differences were observed to another entry blocker, soluble CD4, or to reverse transcriptase, protease, or integrase inhibitors. Chronic as compared to early phase variants demonstrated greater replication when passaged in the presence of subinhibitory concentrations of fusion but not CCR5 receptor inhibitors. Fusion antagonist resistance, however, emerged from only one chronic phase virus culture. Because sensitivity to receptor and fusion antagonists is correlated with receptor affinity and fusion capacity, respectively, changes that occur in the envelope glycoprotein over the course of infection confer greater ability to use the CCR5 receptor and increased fusion ability. Our in vitro passage studies suggest that these evolving phenotypes increase the likelihood of resistance against fusion but not CCR5 receptor blockers. PMID:22650962

The successes and failures of HIV drug discovery.

PubMed

Hashimoto, Chie; Tanaka, Tomohiro; Narumi, Tetsuo; Nomura, Wataru; Tamamura, Hirokazu

2011-10-01

To date, several anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have been developed and used clinically for the treatment of patients infected with HIV. Recently, novel drugs have been discovered which have different mechanisms of action from those of the above inhibitors, including entry inhibitors and integrase (IN) inhibitors; the clinical use of three of these inhibitors has been approved. Other inhibitors are still in development. This review article summarizes the history of the development of anti-HIV drugs and also focuses on successes in the development of these entry and IN inhibitors, along with looking at exploratory approaches for the development of other inhibitors. Currently used highly active antiretroviral therapy can be subject to a loss of efficacy, due to the emergence of multi-drug resistant (MDR) strains; a change of regimens of the drug combination is required to combat this, along with careful monitoring of the virus and CD4 in the blood, by methods such as cellular tropism testing. In such a situation, entry inhibitors such as CCR5/CXCR4 antagonists, CD4 mimics, fusion inhibitors and IN inhibitors might be optional agents for an expansion of the drug repertoire available to patients at all stages of HIV infection.

Identification and characterization of a LTR retrotransposon from the genome of Cyprinus carpio var. Jian.

PubMed

Cao, Liping; Yin, Guojun; Cao, Zheming; Bing, Xuwen; Ding, Weidong

2016-06-01

A Ty3/gypsy-retrotransposon-type transposon was found in the genome of the Jian carp (Cyprinus carpio var. Jian) in a previous study (unpublished), and was designated a JRE retrotransposon (Jian retrotransposon). The full-length JRE retrotransposon is 5126 bp, which includes two long terminal repeats of 470 bp at the 5' end and 453 bp at the 3' end, and two open reading frames between them: 4203 bp encoding the group-specific antigen (GAG) and polyprotein (POL). The pol gene has a typical Ty3/gypsy retrotransposon structure, and the gene order is protease, reverse transcriptase, RNase H, and integrase (PR-RT-RH-IN). A phylogenetic analysis of the pol gene showed that it has similarities of 40.7, 40, and 32.8 %, to retrotransposons of Azumapecten farreri, Mizuhopecten yessoensis, and Xiphophorus maculatus, respectively. Therefore, JRE might belong to the JULE retrotransposon family. The copy number of the JRE transposon in the genome of the Jian carp is 124, determined with real-time quantitative PCR. The mRNA of the JRE retrotransposon is expressed in five Jian carp tissues, the liver, kidney, blood, muscle, and gonad, and slightly higher in the kidney and liver than in the other tissues.

The molecular epidemiology of HIV-1 in the Comunidad Valenciana (Spain): analysis of transmission clusters.

PubMed

Patiño-Galindo, Juan Ángel; Torres-Puente, Manoli; Bracho, María Alma; Alastrué, Ignacio; Juan, Amparo; Navarro, David; Galindo, María José; Ocete, Dolores; Ortega, Enrique; Gimeno, Concepción; Belda, Josefina; Domínguez, Victoria; Moreno, Rosario; González-Candelas, Fernando

2017-09-14

HIV infections are still a very serious concern for public heath worldwide. We have applied molecular evolution methods to study the HIV-1 epidemics in the Comunidad Valenciana (CV, Spain) from a public health surveillance perspective. For this, we analysed 1804 HIV-1 sequences comprising protease and reverse transcriptase (PR/RT) coding regions, sampled between 2004 and 2014. These sequences were subtyped and subjected to phylogenetic analyses in order to detect transmission clusters. In addition, univariate and multinomial comparisons were performed to detect epidemiological differences between HIV-1 subtypes, and risk groups. The HIV epidemic in the CV is dominated by subtype B infections among local men who have sex with men (MSM). 270 transmission clusters were identified (>57% of the dataset), 12 of which included ≥10 patients; 11 of subtype B (9 affecting MSMs) and one (n = 21) of CRF14, affecting predominately intravenous drug users (IDUs). Dated phylogenies revealed these large clusters to have originated from the mid-80s to the early 00 s. Subtype B is more likely to form transmission clusters than non-B variants and MSMs to cluster than other risk groups. Multinomial analyses revealed an association between non-B variants, which are not established in the local population yet, and different foreign groups.

HIV-1 genetic diversity and primary drug resistance mutations before large-scale access to antiretroviral therapy, Republic of Congo.

PubMed

Niama, Fabien Roch; Vidal, Nicole; Diop-Ndiaye, Halimatou; Nguimbi, Etienne; Ahombo, Gabriel; Diakabana, Philippe; Bayonne Kombo, Édith Sophie; Mayengue, Pembe Issamou; Kobawila, Simon-Charles; Parra, Henri Joseph; Toure-Kane, Coumba

2017-07-05

In this work, we investigated the genetic diversity of HIV-1 and the presence of mutations conferring antiretroviral drug resistance in 50 drug-naïve infected persons in the Republic of Congo (RoC). Samples were obtained before large-scale access to HAART in 2002 and 2004. To assess the HIV-1 genetic recombination, the sequencing of the pol gene encoding a protease and partial reverse transcriptase was performed and analyzed with updated references, including newly characterized CRFs. The assessment of drug resistance was conducted according to the WHO protocol. Among the 50 samples analyzed for the pol gene, 50% were classified as intersubtype recombinants, charring complex structures inside the pol fragment. Five samples could not be classified (noted U). The most prevalent subtypes were G with 10 isolates and D with 11 isolates. One isolate of A, J, H, CRF05, CRF18 and CRF37 were also found. Two samples (4%) harboring the mutations M230L and Y181C associated with the TAMs M41L and T215Y, respectively, were found. This first study in the RoC, based on WHO classification, shows that the threshold of transmitted drug resistance before large-scale access to antiretroviral therapy is 4%.

Molecular epidemiology of early and acute HIV type 1 infections in the United States Navy and Marine Corps, 2005-2010.

PubMed

Heipertz, Richard A; Sanders-Buell, Eric; Kijak, Gustavo; Howell, Shana; Lazzaro, Michelle; Jagodzinski, Linda L; Eggleston, John; Peel, Sheila; Malia, Jennifer; Armstrong, Adam; Michael, Nelson L; Kim, Jerome H; O'Connell, Robert J; Scott, Paul T; Brett-Major, David M; Tovanabutra, Sodsai

2013-10-01

The U.S. military represents a unique population within the human immunodeficiency virus 1 (HIV-1) pandemic. The last comprehensive study of HIV-1 in members of the U.S. Navy and Marine Corps (Sea Services) was completed in 2000, before large-scale combat operations were taking place. Here, we present molecular characterization of HIV-1 from 40 Sea Services personnel who were identified during their seroconversion window and initially classified as HIV-1 negative during screening. Protease/reverse transcriptase (pro/rt) and envelope (env) sequences were obtained from each member of the cohort. Phylogenetic analyses were carried out on these regions to determine relatedness within the cohort and calculate the most recent common ancestor for the related sequences. We identified 39 individuals infected with subtype B and one infected with CRF01_AE. Comparison of the pairwise genetic distance of Sea Service sequences and reference sequences in the env and pro/rt regions showed that five samples were part of molecular clusters, a group of two and a group of three, confirmed by single genome amplification. Real-time molecular monitoring of new HIV-1 acquisitions in the Sea Services may have a role in facilitating public health interventions at sites where related HIV-1 infections are identified.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

PubMed

Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

2016-05-01

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

TNF α is involved in neuropathic pain induced by nucleoside reverse transcriptase inhibitor in rats

PubMed Central

Zheng, Xuexing; Ouyang, Handong; Liu, Shue; Mata, Marina; Fink, David J.; Hao, Shuanglin

2011-01-01

In patients with HIV/AIDS, neuropathic pain is a common neurological complication. Infection with the HIV itself may lead to neuropathic pain, and painful symptoms are enhanced when patients are treated with nucleoside reverse transcriptase inhibitors (NRTI). The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current studies, we tested the role of TNFα in antiretroviral drug-induced neuropathic pain. We administered 2′,3′-dideoxycytidine (ddC, one of the NRTIs) systemically to induce mechanical allodynia. We found that ddC induced overexpression of both mRNA and proteins of GFAP and TNFα in the spinal dorsal horn. TNFα was colocalized with GFAP in the spinal dorsal horn and with NeuN in the DRG. Knockdown of TNFα with siRNA blocked the mechanical allodynia induced by ddC. Intrathecal administration of glial inhibitor or recombinant TNF soluble receptor, reversed mechanical allodynia induced by ddC. These results suggest that TNFα is involved in NRTI-induced neuropathic pain. PMID:21741472

Crystallographic Study of a Novel Sub-Nanomolar Inhibitor Provides Insight on the Binding Interactions of Alkenyldiarylmethanes with Human Immunodeficiency Virus-1 (HIV-1) Reverse Transcriptase†

PubMed Central

Cullen, Matthew D.; Ho, William C.; Bauman, Joseph D.; Das, Kalyan; Arnold, Eddy; Hartman, Tracy L.; Watson, Karen M.; Buckheit, Robert W.; Pannecouque, Christophe; De Clercq, Erik; Cushman, Mark

2009-01-01

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the backside of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC50 ≤ nM). ADAM 4 was designed to be hydrolytically stable in blood plasma, and an investigation of its hydrolysis in rat plasma demonstrated it has a significantly prolonged half-life in comparison to ADAM lead compounds 1 and 2. PMID:19775161

Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy.

PubMed

Lewis, William

2003-01-01

Nucleoside reverse-transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are the cornerstones of current AIDS therapy, but extensive use brought mitochondrial side effects to light. Clinical experience, pharmacological, cell, and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in target tissues are observed. Organ-specific pathological changes or diverse systemic effects result from and are frequently attributed to HAART in which NRTIs are included. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself. Copyright 2003, Elsevier Science (USA)

An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

PubMed

He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

2009-06-01

Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo.

An Intravaginal Ring That Releases the NNRTI MIV-150 Reduces SHIV Transmission in Macaques

PubMed Central

Rodriguez, Aixa; Kizima, Larisa; Menon, Radhika; Goldman, Daniel; Kenney, Jessica; Aravantinou, Meropi; Seidor, Samantha; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Fernández-Romero, José A.; Robbiani, Melissa; Zydowsky, Thomas M.

2015-01-01

Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150–containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs. PMID:22956201

An intravaginal ring that releases the NNRTI MIV-150 reduces SHIV transmission in macaques.

PubMed

Singer, Rachel; Mawson, Paul; Derby, Nina; Rodriguez, Aixa; Kizima, Larisa; Menon, Radhika; Goldman, Daniel; Kenney, Jessica; Aravantinou, Meropi; Seidor, Samantha; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, José A; Robbiani, Melissa; Zydowsky, Thomas M

2012-09-05

Microbicides may prevent HIV and sexually transmitted infections (STIs) in women; however, determining the optimal means of delivery of active pharmaceutical ingredients remains a major challenge. We previously demonstrated that a vaginal gel containing the non-nucleoside reverse transcriptase inhibitor MIV-150 partially protected macaques from SHIV-RT (simian/HIV reverse transcriptase) infection, and the addition of zinc acetate rendered the gel significantly protective. We test the activity of MIV-150 without the addition of zinc acetate when delivered from either ethylene vinyl acetate (EVA) or silicone intravaginal rings (IVRs). MIV-150 was successfully delivered, because it was detected in vaginal fluids and tissues by radioimmunoassay in pharmacokinetic studies. Moreover, EVA IVRs significantly protected macaques from SHIV-RT infection. Our results demonstrate that MIV-150-containing IVRs have the potential to prevent HIV infection and highlight the possible use of IVRs for delivering drugs that block HIV and other STIs.

Practical diagnostic testing for human immunodeficiency virus.

PubMed Central

Jackson, J B; Balfour, H H

1988-01-01

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures. Images PMID:3060241

Telomerase Mechanism of Telomere Synthesis

PubMed Central

Wu, R. Alex; Upton, Heather E.; Vogan, Jacob M.; Collins, Kathleen

2017-01-01

Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines. PMID:28141967

Human telomerase reverse transcriptase is a promising target for cancer inhibition in squamous cell carcinomas.

PubMed

Park, Young-Jin; Kim, Eun-Kyoung; Moon, Sook; Hong, Doo-Pyo; Bae, Jung Yoon; Kim, Jin

2014-11-01

The present study aimed to investigate whether the down-regulation of human telomerase reverse transcriptase (hTERT) may induce an anti-invasive effect in oral squamous cell cancer cell lines. A genetically-engineered squamous carcinoma cell line overexpressing hTERT in immortalized oral keratinocytes transfected by human papilloma virus (HPV)-16 E6/E7 (IHOK) was used. In vivo tumorigenicity was examined using an orthotopic xenograft model of nude mice. For evaluating anti-invasive activity by knockdown of hTERT expression, transwell invasion assay and real-time polymerase chain reaction (PCR) for matrix metalloproteinases (MMP) were employed. The down-regulation of hTERT expression reduced the invasive activity and MMP expression. This result was re-confirmed in the HSC3 oral squamous carcinoma cell line. Targeting hTERT may lead to novel therapeutic approaches. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate

NASA Technical Reports Server (NTRS)

Setlik, R. F.; Meyer, D. J.; Shibata, M.; Roskwitalski, R.; Ornstein, R. L.; Rein, R.

1994-01-01

We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy.

PubMed

Crawford, Keith W; Njeru, Dorothy; Maswai, Jonah; Omondi, Milton; Apollo, Duncan; Kimetto, Jane; Gitonga, Lawrence; Munyao, James; Langat, Raphael; Aoko, Appolonia; Tarus, Jemutai; Khamadi, Samoel; Hamm, Tiffany E

2014-01-28

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

PubMed

Tang, Jing; Vernekar, Sanjeev Kumar V; Chen, Yue-Lei; Miller, Lena; Huber, Andrew D; Myshakina, Nataliya; Sarafianos, Stefan G; Parniak, Michael A; Wang, Zhengqiang

2017-06-16

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Transposable elements in sexual and ancient asexual taxa

PubMed Central

Arkhipova, Irina; Meselson, Matthew

2000-01-01

Sexual reproduction allows deleterious transposable elements to proliferate in populations, whereas the loss of sex, by preventing their spread, has been predicted eventually to result in a population free of such elements [Hickey, D. A. (1982) Genetics 101, 519–531]. We tested this expectation by screening representatives of a majority of animal phyla for LINE-like and gypsy-like reverse transcriptases and mariner/Tc1-like transposases. All species tested positive for reverse transcriptases except rotifers of the class Bdelloidea, the largest eukaryotic taxon in which males, hermaphrodites, and meiosis are unknown and for which ancient asexuality is supported by molecular genetic evidence. Mariner-like transposases are distributed sporadically among species and are present in bdelloid rotifers. The remarkable lack of LINE-like and gypsy-like retrotransposons in bdelloids and their ubiquitous presence in other taxa support the view that eukaryotic retrotransposons are sexually transmitted nuclear parasites and that bdelloid rotifers evolved asexually. PMID:11121049

Induced maturation of human immunodeficiency virus.

PubMed

Mattei, Simone; Anders, Maria; Konvalinka, Jan; Kräusslich, Hans-Georg; Briggs, John A G; Müller, Barbara

2014-12-01

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

PubMed

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Molecular docking of (5E)-3-(2-aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione on HIV-1 reverse transcriptase: novel drug acting on enzyme.

PubMed

Seniya, Chandrabhan; Yadav, Ajay; Uchadia, Kuldeep; Kumar, Sanjay; Sagar, Nitin; Shrivastava, Priyanka; Shrivastava, Shilpi; Wadhwa, Gulshan

2012-01-01

The study of Human immunodeficiency virus (HIV) in humans and animal models in last 31 years suggested that it is a causative agent of AIDS. This causes serious pandemic public health concern globally. It was reported that the HIV-1 reverse transcriptase (RT) played a critical role in the life cycle of HIV. Therefore, inhibition of HIV-1RT enzyme is one of the major and potential targets in the treatment of AIDS. The enzyme (HIV-1RT) was successfully targeted by non nucleotide reverse transcriptase inhibitors (NNRTIs). But frequent application of NNRTIs led drug resistance mutation on HIV infections. Therefore, there is a need to search new NNRTIs with appropriate pharmacophores. For the purpose, a virtually screened 3D model of unliganded HIV-1RT (1DLO) was explored. The unliganded HIV-1RT (1DLO) was docked with 4-thiazolidinone and its derivatives (ChemBank Database) by using AutoDock4. The best seven docking solutions complex were selected and analyzed by Ligplot. The analysis showed that derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) has maximum potential against unliganded HIV-1RT (1DLO). The analysis was done on the basis of scoring and binding ability. The derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) indicated minimum energy score and highest number of interactions with active site residue and could be a promising inhibitor for HIV-1 RT as Drug target.

Screening for diverse PDGFRA or PDGFRB fusion genes is facilitated by generic quantitative reverse transcriptase polymerase chain reaction analysis

PubMed Central

Erben, Philipp; Gosenca, Darko; Müller, Martin C.; Reinhard, Jelena; Score, Joannah; del Valle, Francesco; Walz, Christoph; Mix, Jürgen; Metzgeroth, Georgia; Ernst, Thomas; Haferlach, Claudia; Cross, Nicholas C.P.; Hochhaus, Andreas; Reiter, Andreas

2010-01-01

Background Rapid identification of diverse fusion genes with involvement of PDGFRA or PDGFRB in eosinophilia-associated myeloproliferative neoplasms is essential for adequate clinical management but is complicated by the multitude and heterogeneity of partner genes and breakpoints. Design and Methods We established a generic quantitative reverse transcriptase polymerase chain reaction to detect overexpression of the 3′-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion. Results At diagnosis, all patients with known fusion genes involving PDGFRA (n=5; 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 versus 0.0066 versus 0.0064, P<0.0001; PDGFRB/ABL: 196 versus 3.8 versus 5.85, P<0.0001). The sensitivity and specificity of the activation screening test were, respectively, 100% and 88.4% for PDGFRA and 100% and 94% for PDGFRB. Furthermore, significant overexpression of PDGFRB was found in a patient with an eosinophilia-associated myeloproliferative neoplasm with uninformative cytogenetics and an excellent response to imatinib. Subsequently, a new SART3-PDGFRB fusion gene was identified by 5′-rapid amplification of cDNA ends polymerase chain reaction (5′-RACE-PCR). Conclusions Quantitative reverse transcriptase polymerase chain reaction analysis is a simple and useful adjunct to standard diagnostic assays to detect clinically significant overexpression of PDGFRA and PDGFRB in eosinophilia-associated myeloproliferative neoplasms or related disorders. PMID:20107158

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PubMed

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S; Moncla, Bernard; Sarafianos, Stefan G; Parniak, Michael A; Rohan, Lisa C

2015-09-01

EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

Enzyme processing of textiles in reverse micellar solution.

PubMed

Sawada, K; Ueda, M

2001-08-23

Scouring of cotton using pectinase enzyme, bioscouring, in reverse micellar system was studied. The effectiveness of bioscouring was evaluated by measuring weight loss of cotton, analyzing pectin and cotton wax remaining and by wetness testing. Pectinase enzyme showed excellent activity even in organic media, and the effectiveness of scouring was equivalent or better than that achieved by conventional alkaline process or bioscouring in aqueous media. Enzymatic modification of wool using protease enzyme in the same system was also studied. It has found that felting property and tensile strength of wool fabrics treated by protease in reverse micellar system were superior to those in aqueous media. Possibilities of utilization of the same system for the subsequent textile dyeing process were also investigated. It was found that cotton and polyester fabrics were dyed satisfactorily by reverse micellar system compared to conventional aqueous system.

TELOMERASE AND CHRONIC ARSENIC EXPOSURE IN HUMANS

EPA Science Inventory

Arsenic exposure has been associated with increased risk of skin, lung and bladder cancer in humans. The mechanisms of carcinogenesis are not well understood. Telomerase, a ribonucleoprotein containing human telomerase reverse transcriptase (hTERT), can extend telomeres of eukary...

A reverse transcriptase-dependent mechanism plays central roles in fundamental biological processes.

PubMed

Spadafora, Corrado

2008-01-01

This review summarizes emerging evidence that LINE-1 (Long Interspersed Nuclear Elements) -encoded reverse transcriptase (RT) regulates fundamental biological processes. Earlier studies showed that sperm cells can be used as vectors of both exogenous DNA and RNA molecules in sperm-mediated gene transfer assays. During these studies, a sperm endogenous RT activity was identified, which can reverse-transcribe exogenous RNA directly, or DNA molecules through sequential transcription and reverse transcription. Resulting cDNA copies generated in sperm cells can be delivered to embryos at fertilization, further propagated in tissues as low-copy extrachromosomal structures and transmitted to the progeny in a non-mendelian fashion. Being transcriptionally competent, they can induce phenotypic variations in positive tissues. An RT activity is also present in preimplantation embryos, and its inhibition causes developmental arrest in early preimplantation stages, paralleled by an extensive reprogramming of gene expression. In analogy with this, drug-mediated inhibition of RT activity, or RNA interference-mediated silencing of human LINE-1, reduce cell proliferation and induce differentiation in a variety of cancer cell lines. Furthermore, RT inhibition in vivo antagonizes the growth of human tumors in animal models. As a whole, these data implicate a RT-dependent machinery in the genesis of new genetic information in spermatozoa and in normal and pathological developmental processes.

Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

PubMed Central

Castagliuolo, Ignazio; Riegler, Martin F.; Valenick, Leyla; LaMont, J. Thomas; Pothoulakis, Charalabos

1999-01-01

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease. PMID:9864230

Applications of 2D IR spectroscopy to peptides, proteins, and hydrogen-bond dynamics

PubMed Central

Kim, Yung Sam; Hochstrasser, Robin M.

2010-01-01

Following a survey of 2D IR principles this Feature Article describes recent experiments on the hydrogen-bond dynamics of small ions, amide-I modes, nitrile probes, peptides, reverse transcriptase inhibitors, and amyloid fibrils. PMID:19351162

Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys

EPA Science Inventory

Cryptosporidium spp. and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (...

Epigenetic Characterization of Ovarian Cancer

DTIC Science & Technology

2008-12-01

Gusberg, A. H., Whitaker, R. S., Gray , J. W., Fujii, S., Berchuck, A. and S. K. Murphy. YY1/E2F3 modulates antimicrotubule drug response in epithelial... GTG GGT TTT TGG TGT TGG GTA TT-3’; and a shared reverse primer that does not anneal to CpGs, 5’-AAC CCC ACT CCC ACC CTA CTC C-3’. PCR was performed...Superscript II RNase H- reverse transcriptase (Invitrogen). Forward primer: 5’-GCG ACA TCG GTG ACT TCA T-3’ and reverse primer 5’-ATA CAT GTC CGC CAG CTT

Increasingly successful highly active antiretroviral therapy delays the emergence of new HLA class I-associated escape mutations in HIV-1.

PubMed

Knapp, David J H F; Brumme, Zabrina L; Huang, Sheng Yuan; Wynhoven, Brian; Dong, Winnie W Y; Mo, Theresa; Harrigan, P Richard; Brumme, Chanson J

2012-06-01

HLA class I-restricted cytotoxic T lymphocytes and highly active antiretroviral therapy (HAART) exert strong selective pressures on human immunodeficiency virus type 1 (HIV-1), leading to escape mutations compromising virologic control. Immune responses continue to shape HIV-1 evolution after HAART initiation, but the extent and rate at which this occurs remain incompletely quantified. Here, we characterize the incidence and clinical correlates of HLA-associated evolution in HIV-1 Pol after HAART initiation in a large, population-based observational cohort. British Columbia HAART Observational, Medical Evaluation and Research cohort participants with available HLA class I types and longitudinal posttherapy protease/reverse transcriptase sequences were studied (n = 619; median, 5 samples per patient and 5.2 years of follow-up). HLA-associated polymorphisms were defined according to published reference lists. Rates and correlates of immune-mediated HIV-1 evolution were investigated using multivariate Cox proportional hazard models incorporating baseline and time-dependent plasma viral load and CD4 response data. New HLA-associated escape events were observed in 269 (43%) patients during HAART and occurred at 49 of 63 (78%) investigated immune-associated sites in Pol. In time-dependent analyses adjusting for baseline factors, poorer virologic, but not immunologic, response to HAART was associated with increased risk of immune escape of 1.9-fold per log(10) viral load increment (P < .0001). Reversion of escape mutations following HAART initiation was extremely rare. HLA-associated HIV-1 evolution continues during HAART to an extent that is inversely related to the virologic success of therapy. Minimizing the degree of immune escape could represent a secondary benefit of effective HAART.

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

DOE PAGES

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.; ...

2017-07-05

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

HPLC-MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long term stability in different conditions.

PubMed

D'Avolio, Antonio; Simiele, Marco; Siccardi, Marco; Baietto, Lorena; Sciandra, Mauro; Bonora, Stefano; Di Perri, Giovanni

2010-09-05

A bioanalytical method for the determination of most commonly prescribed protease inhibitors (saquinavir, atazanavir, amprenavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (etravirine, efavirenz and nevirapine) was developed, modifying our previous HPLC-MS chromatographic run, validated and a complete short and long term stability evaluation was carried out. One hundred microlitres of plasma were distributed on a collection glass paper filter (Glass-Microfibre from Sartorius), then the filter underwent thermal treatment, both for drying and for HIV inactivation, and stored at room temperature, 4 degrees C and -20 degrees C. The analytes were extracted from the filter disc using tert-butylmethylether with basic pH, after the addition of the internal standards quinoxaline. The extract was dried, reconstituted and the chromatographic separation was performed on a reversed-phase C-18 column (150 mm x 2.0 mm) and the analytes were quantified using a single quadrupole mass spectrometer. The method was validated considering the concentration ranges encountered in clinical trials and the routine clinical practice. The assay was linear over the concentration ranges tested. Accuracies ranged from 92.1% to 111.9% and intra-day and inter-day relative standard deviation for all quality control levels ranged from 0.2 to 12.9 and 3.1 to 14.4, respectively. Analytes in dried plasma spots were stable for longer time when dried/inactivation step was carried out before storage compared to samples not dried/inactivated before the analysis. The dried/inactivation step allows shipment of samples at room temperature without any risks, therefore the developed and validated method enables an easy and cheap sample shipment for therapeutic drug monitoring and pharmacokinetic studies. 2010 Elsevier B.V. All rights reserved.

Theoretical benefits of mitogen applications for HIV-1 infections.

PubMed

Wimer, B M; Morris, R E

1997-06-01

Ideal treatment of HIV-1 infections should include an agent that can reverse the capacity of the virus to evade destruction by hiding in sanctuaries and by frequently mutating the epitopes it displays. The rapid proliferation of virions during the years of symptomatic quiescence obligates rapid replacement of CD4+ lymphocytes that leads to a gradual attrition of the T lymphocytes needed to control infections. In vitro evidences suggest that, given systematically, certain mitogenic lectins would interfere with HIV-1 invasion of CD4+ cells by blocking gp120 molecules on the viral membrane before activating T lymphocytes subsequent to binding with their Ti/CD3 molecules. The nonspecific nature of antiviral effector cells generated by this activation should circumvent HIV-1 mutations at the same time it reconstitutes depleted T lymphocytes, stimulates myelopoiesis, and reinforces resistance to malignancies and infections prevalent with the immunodeficiency state. Properly coordinating these effects with appropriate combinations of reverse transcriptase and protease inhibitors could theoretically expedite complete elimination of HIV in a timely fashion that shorten the required treatment duration and excludes the detrimental effects of virus mutations. The proper sequence of this treatment should be maximum reduction of the HIV-1 load with drug combinations, control of complicating infection by other means to reduce mitogen-induced tissue necrosis, and addition of systemic PHA-L4 administration regulated to maintain a 5-10 micrograms/mL serum concentration. The antiviral regimen should be continued an undetermined time beyond when HIV-1 is no longer detectable, and systemic L4 administration until satisfactory immunologic and hematologic competences are re-established. Partially-matched mitogen-activated adoptive leukocyte therapy might be additionally helpful.

Problem-Solving Test: Catalytic Activities of a Human Nuclear Enzyme

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: ion exchange chromatography, polynucleotides, oligonucleotides, radioactive labeling, template, primer, DNA polymerase, reverse transcriptase, helicase, nucleoside triphosphates, nucleoside diphosphates, nucleoside monophosphates, nucleosides, 5'-end and 3'-end, bacteriophage,…

Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005. 11(8): 1303–1305)

EPA Pesticide Factsheets

Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, aerosol, liquid and water samples. This is a two-step, real-time reverse transcriptase-PCR multiplex assay.

Liver toxicity associated with antiretroviral therapy including efavirenz or ritonavir-boosted protease inhibitors in a cohort of HIV/hepatitis C virus co-infected patients.

PubMed

Neukam, Karin; Mira, José A; Ruiz-Morales, Josefa; Rivero, Antonio; Collado, Antonio; Torres-Cornejo, Almudena; Merino, Dolores; de Los Santos-Gil, Ignacio; Macías, Juan; González-Serrano, Mercedes; Camacho, Angela; Parra-García, Ginés; Pineda, Juan A

2011-11-01

To compare the frequency of grade 3 or 4 transaminase elevations (TEs) in HIV/hepatitis C virus (HCV) co-infected patients who started a three-antiretroviral drug regimen including efavirenz or a ritonavir-boosted protease inhibitor (PI/r) and the influence of pre-existing significant hepatic fibrosis or cirrhosis. All pre-treated or treatment-naive HIV/HCV co-infected patients who started an antiretroviral regimen including two nucleos(t)ide reverse transcriptase inhibitors along with efavirenz or a PI/r in seven Spanish centres from January 2007 to December 2009 were included in this prospective study. Of 262 patients included in this study, 76 (29%) individuals began antiretroviral therapy (ART) including efavirenz and 186 (71%) a PI/r-based combination. The median (interquartile) follow-up was 14.0 (6.2-23.7) months. A total of 20 (7.6%) patients presented grade 3-4 TEs. Four (1.5%) subjects discontinued ART due to this adverse event. Grade 3-4 TEs were observed in 5 (6.6%) subjects receiving efavirenz and 15 (8.1%) treated with PI/r (P = 0.681). Three (6.5%) patients in the efavirenz group with significant fibrosis developed grade 3-4 TEs versus 2 (8.7%) without pre-existing significant fibrosis (P = 0.743). In the PI/r group, the corresponding figures were 10 (8.8%) and 5 (9.3%), respectively (P = 0.931). The frequency of grade 3-4 TEs associated with efavirenz-based ART combinations under clinical practice conditions is low and similar to that found in patients receiving PI/r currently used in HIV/HCV co-infected patients. The baseline fibrosis stage does not have an impact on the development of TEs caused by these antiretroviral drugs in this population.

A cross-sectional study to evaluate the association of hyperbilirubinaemia on markers of cardiovascular disease, neurocognitive function, bone mineral density and renal markers in HIV-1 infected subjects on protease inhibitors.

PubMed

Barber, T J; Moyle, G; Hill, A; Jagjit Singh, G; Scourfield, A; Yapa, H M; Waters, L; Asboe, D; Boffito, M; Nelson, M

2016-05-01

Ongoing inflammation in controlled HIV infection contributes to non-AIDS comorbidities. High bilirubin appears to exhibit an anti-inflammatory effect in vivo. We therefore examined whether increased bilirubin in persons with HIV was associated with differences in markers of inflammation and cardiovascular, bone, renal disease, and neurocognitive (NC) impairment. This cross-sectional study examined inflammatory markers in individuals with stable HIV infection treated with two nucleoside reverse transcriptase inhibitors and a boosted protease inhibitor. Individuals recruited were those with a normal bilirubin (NBR; 0-17 μmol/L) or high bilirubin (>2.5 × upper limit of normal). Demographic and anthropological data were recorded. Blood and urine samples were taken for analyses. Pulse wave velocity (PWV) measurement, carotid intimal thickness (CIT), and calcaneal stiffness (CSI) were measured. Males were asked to answer a questionnaire about sexual function; NC testing was performed using CogState. 101 patients were screened, 78 enrolled (43 NBR and 35 HBR). Atazanavir use was significantly higher in HBR. Whilst a trend for lower CIT was seen in those with HBR, no significant differences were seen in PWV, bone markers, calculated cardiovascular risk (Framingham), or erectile dysfunction score. VCAM-1 levels were significantly lower in the HBR group. HBR was associated with lower LDL and triglyceride levels. NBR was associated with a calculated FRAX significantly lower than HBR although no associations were found after adjusting for tenofovir use. No difference in renal markers was observed. Component tests of NC testing revealed differences favouring HBR but overall composite scores were similar. High bilirubin in the context of boosted PI therapy was found not to be associated with differences in with the markers examined in this study. Some trends were noted and, on the basis of these, a larger, clinical end point study is warranted.

Secondary structure model of the RNA recognized by the reverse transcriptase from the R2 retrotransposable element.

PubMed Central

Mathews, D H; Banerjee, A R; Luan, D D; Eickbush, T H; Turner, D H

1997-01-01

RNA transcripts corresponding to the 250-nt 3' untranslated region of the R2 non-LTR retrotransposable element are recognized by the R2 reverse transcriptase and are sufficient to serve as templates in the target DNA-primed reverse transcription (TPRT) reaction. The R2 protein encoded by the Bombyx mori R2 can recognize this region from both the B. mori and Drosophila melanogaster R2 elements even though these regions show little nucleotide sequence identity. A model for the RNA secondary structure of the 3' untranslated region of the D. melanogaster R2 retrotransposon was developed by sequence comparison of 10 species aided by free energy minimization. Chemical modification experiments are consistent with this prediction. A secondary structure model for the 3' untranslated region of R2 RNA from the R2 element from B. mori was obtained by a combination of chemical modification data and free energy minimization. These two secondary structure models, found independently, share several common sites. This study shows the utility of combining free energy minimization, sequence comparison, and chemical modification to model an RNA secondary structure. PMID:8990394

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

PubMed

Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

2016-04-01

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Vaginal microbicide film combinations of two reverse transcriptase inhibitors, EFdA and CSIC, for the prevention of HIV-1 sexual transmission

PubMed Central

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S.; Moncla, Bernard; Sarafianos, Stefan G.; Parniak, Michael A.; Rohan, Lisa C.

2015-01-01

Purpose EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Methods Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. Results No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Conclusions Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission. PMID:25794967

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART.

PubMed

Nouchi, A; Nguyen, T; Valantin, M A; Simon, A; Sayon, S; Agher, R; Calvez, V; Katlama, C; Marcelin, A G; Soulie, C

2018-05-29

To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

PubMed

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R; Shell, Elisabeth J; Parkin, Neil; O'Brien, William A; Ferguson, Monique R

2008-10-01

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility

PubMed Central

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R.; Shell, Elisabeth J.; Parkin, Neil; O'Brien, William A.

2008-01-01

Abstract The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals. PMID:18844463

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

PubMed

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

PubMed Central

Burke, W D; Calalang, C C; Eickbush, T H

1987-01-01

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover. Images PMID:2439905

Giant Reverse Transcriptase-Encoding Transposable Elements at Telomeres.

PubMed

Arkhipova, Irina R; Yushenova, Irina A; Rodriguez, Fernando

2017-09-01

Transposable elements are omnipresent in eukaryotic genomes and have a profound impact on chromosome structure, function and evolution. Their structural and functional diversity is thought to be reasonably well-understood, especially in retroelements, which transpose via an RNA intermediate copied into cDNA by the element-encoded reverse transcriptase, and are characterized by a compact structure. Here, we report a novel type of expandable eukaryotic retroelements, which we call Terminons. These elements can attach to G-rich telomeric repeat overhangs at the chromosome ends, in a process apparently facilitated by complementary C-rich repeats at the 3'-end of the RNA template immediately adjacent to a hammerhead ribozyme motif. Terminon units, which can exceed 40 kb in length, display an unusually complex and diverse structure, and can form very long chains, with host genes often captured between units. As the principal polymerizing component, Terminons contain Athena reverse transcriptases previously described in bdelloid rotifers and belonging to the enigmatic group of Penelope-like elements, but can additionally accumulate multiple cooriented ORFs, including DEDDy 3'-exonucleases, GDSL esterases/lipases, GIY-YIG-like endonucleases, rolling-circle replication initiator (Rep) proteins, and putatively structural ORFs with coiled-coil motifs and transmembrane domains. The extraordinary length and complexity of Terminons and the high degree of interfamily variability in their ORF content challenge the current views on the structural organization of eukaryotic retroelements, and highlight their possible connections with the viral world and the implications for the elevated frequency of gene transfer. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Expression of Connexin 43 in Synovial Tissue of Patients With Rheumatoid Arthritis

PubMed Central

MATSUKI, Tomohiro; TSUCHIDA, Shinji; TERAUCHI, Ryu; ODA, Ryo; FUJIWARA, Hiroyoshi; MAZDA, Osam; KUBO, Toshikazu

2016-01-01

Objectives This study aims to identify the distribution and expression level of connexin 43 (Cx43) in synovial tissue in patients with rheumatoid arthritis (RA). Patients and methods The expression of Cx43 in synovial tissue from eight patients with RA (2 males, 6 females; mean age 59.5±2.7 years; range 52 to 71 years), five patients with osteoarthritis (2 males, 3 females; mean age 68.4±2.7 years; range 61 to 81 years), and one normal female subject (mean age 61 year) was analyzed by quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry of tissue sections. Induction of Cx43 following stimulation of human RA synovial fibroblasts with tumor necrosis factor-alpha (TNF-a) cultures was examined by quantitative reverse transcriptase polymerase chain reaction. The effect of small interfering ribonucleic acid targeting Cx43 (siCx43) on the expression of TNF-a and interleukin-6 was examined using quantitative reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assays. Results Connexin 43 was highly expressed in RA synovial tissue, which also expressed TNF-a, but was expressed lower in osteoarthritis and normal synovial tissue. Expression of Cx43 was markedly up-regulated in RA synovial fibroblasts after stimulation with TNF-a. The over-expression of pro- inflammatory cytokines was suppressed by transfection of siCx43. Conclusion This study shows that Cx43 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNF-a. The expression of the pro-inflammatory cytokines was inhibited by transfection of siCx43. Cx43 may be a novel marker of inflammation in RA synovial tissue. PMID:29900991

Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells.

PubMed

Haendeler, Judith; Hoffmann, Jörg; Diehl, J Florian; Vasa, Mariuca; Spyridopoulos, Ioakim; Zeiher, Andreas M; Dimmeler, Stefanie

2004-04-02

Aging is associated with a rise in intracellular reactive oxygen species (ROS) and a loss of telomerase reverse transcriptase activity. Incubation with H2O2 induced the nuclear export of telomerase reverse transcriptase (TERT) into the cytosol in a Src-family kinase-dependent manner. Therefore, we investigated the hypothesis that age-related increase in reactive oxygen species (ROS) may induce the nuclear export of TERT and contribute to endothelial cell senescence. Continuous cultivation of endothelial cells resulted in an increased endogenous formation of ROS starting after 29 population doublings (PDL). This increase was accompanied by mitochondrial DNA damage and preceded the onset of replicative senescence at PDL 37. Along with the enhanced formation of ROS, we detected an export of nuclear TERT protein from the nucleus into the cytoplasm and an activation of the Src-kinase. Moreover, the induction of premature senescence by low concentrations of H2O2 was completely blocked with the Src-family kinase inhibitor PP2, suggesting a crucial role for Src-family kinases in the induction of endothelial cell aging. Incubation with the antioxidant N-acetylcysteine, from PDL 26, reduced the intracellular ROS formation and prevented mitochondrial DNA damage. Likewise, nuclear export of TERT protein, loss in the overall TERT activity, and the onset of replicative senescence were delayed by incubation with N-acetylcysteine. Low doses of the statin, atorvastatin (0.1 micromol/L), had also effects similar to those of N-acetylcysteine. We conclude that both antioxidants and statins can delay the onset of replicative senescence by counteracting the increased ROS production linked to aging of endothelial cells.

Potent NLRP3 Inflammasome Activation by the HIV Reverse Transcriptase Inhibitor Abacavir.

PubMed

Toksoy, Atiye; Sennefelder, Helga; Adam, Christian; Hofmann, Sonja; Trautmann, Axel; Goebeler, Matthias; Schmidt, Marc

2017-02-17

There is experimental and clinical evidence that some exanthematous allergic drug hypersensitivity reactions are mediated by drug-specific T cells. We hypothesized that the capacity of certain drugs to directly stimulate the innate immune system may contribute to generate drug-specific T cells. Here we analyzed whether abacavir, an HIV-1 reverse transcriptase inhibitor often inducing severe delayed-type drug hypersensitivity, can trigger innate immune activation that may contribute to its allergic potential. We show that abacavir fails to generate direct innate immune activation in human monocytes but potently triggers IL-1β release upon pro-inflammatory priming with phorbol ester or Toll-like receptor stimulation. IL-1β processing and secretion were sensitive to Caspase-1 inhibition, NLRP3 knockdown, and K + efflux inhibition and were not observed with other non-allergenic nucleoside reverse transcriptase inhibitors, identifying abacavir as a specific inflammasome activator. It further correlated with dose-dependent mitochondrial reactive oxygen species production and cytotoxicity, indicating that inflammasome activation resulted from mitochondrial damage. However, both NLRP3 depletion and inhibition of K + efflux mitigated abacavir-induced mitochondrial reactive oxygen species production and cytotoxicity, suggesting that these processes were secondary to NLRP3 activation. Instead, depletion of cardiolipin synthase 1 abolished abacavir-induced IL-1β secretion, suggesting that mitochondrial cardiolipin release may trigger abacavir-induced inflammasome activation. Our data identify abacavir as a novel inflammasome-stimulating drug allergen. They implicate a potential contribution of innate immune activation to medication-induced delayed-type hypersensitivity, which may stimulate new concepts for treatment and prevention of drug allergies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative analysis of drug resistance mutations in the human immunodeficiency virus reverse transcriptase gene in patients who are non-responsive, responsive and naive to antiretroviral therapy.

PubMed

Misbah, Mohammad; Roy, Gaurav; Shahid, Mudassar; Nag, Nalin; Kumar, Suresh; Husain, Mohammad

2016-05-01

Drug resistance mutations in the Pol gene of human immunodeficiency virus 1 (HIV-1) are one of the critical factors associated with antiretroviral therapy (ART) failure in HIV-1 patients. The issue of resistance to reverse transcriptase inhibitors (RTIs) in HIV infection has not been adequately addressed in the Indian subcontinent. We compared HIV-1 reverse transcriptase (RT) gene sequences to identify mutations present in HIV-1 patients who were ART non-responders, ART responders and drug naive. Genotypic drug resistance testing was performed by sequencing a 655-bp region of the RT gene from 102 HIV-1 patients, consisting of 30 ART-non-responding, 35 ART-responding and 37 drug-naive patients. The Stanford HIV Resistance Database (HIVDBv 6.2), IAS-USA mutation list, ANRS_09/2012 algorithm, and Rega v8.02 algorithm were used to interpret the pattern of drug resistance. The majority of the sequences (96 %) belonged to subtype C, and a few of them (3.9 %) to subtype A1. The frequency of drug resistance mutations observed in ART-non-responding, ART-responding and drug-naive patients was 40.1 %, 10.7 % and 20.58 %, respectively. It was observed that in non-responders, multiple mutations were present in the same patient, while in responders, a single mutation was found. Some of the drug-naive patients had more than one mutation. Thymidine analogue mutations (TAMs), however, were found in non-responders and naive patients but not in responders. Although drug resistance mutations were widely distributed among ART non-responders, the presence of resistance mutations in the viruses of drug-naive patients poses a big concern in the absence of a genotyping resistance test.

RNA–protein binding interface in the telomerase ribonucleoprotein

PubMed Central

Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

2011-01-01

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

In vitro cross-resistance profile of nucleoside reverse transcriptase inhibitor (NRTI) BMS-986001 against known NRTI resistance mutations.

PubMed

Li, Zhufang; Terry, Brian; Olds, William; Protack, Tricia; Deminie, Carol; Minassian, Beatrice; Nowicka-Sans, Beata; Sun, Yongnian; Dicker, Ira; Hwang, Carey; Lataillade, Max; Hanna, George J; Krystal, Mark

2013-11-01

BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.

Therapeutic utility and medicinal chemistry of cathepsin C inhibitors.

PubMed

Guay, Daniel; Beaulieu, Christian; Percival, M David

2010-01-01

The lysosomal cysteine protease cathepsin C (Cat C), also known as dipeptidyl peptidase I, activates a number of granule-associated serine proteases with pro-inflammatory and immune functions by removal of their inhibitory N-terminal dipeptides. Thus, Cat C is a therapeutic target for the treatment of a number of inflammatory and autoimmune diseases. Cathepsin C null mice and humans with Cat C loss of function mutations (Papillon-Lefèvre syndrome) show deficiencies in disease-relevant proteases including neutrophil elastase, cathepsin G, chymases and granzymes and the Cat C mice are protected in a number of disease models. Several methodologies have been recently reported for assessing the effects of Cat C inhibitors on serine protease activities in cellular assays and prolonged treatment of rats with a reversible, selective Cat C inhibitor reduced the activity of three leukocyte serine proteases. Nearly all potent and selective Cat C inhibitors described are based on the preferred dipeptide substrates bearing either irreversible (e.g. diazomethylketone, acyloxymethyl ketone, o-acyl hydroxamic acid and vinyl sulfone) or reversible (e.g. semicarbazide, nitrile and cyanamide) electrophilic warheads. While potent and highly selective, the best inhibitors described to date still have poor stability and/or rodent pharmacokinetics, likely resulting from their peptidic nature. The lack of selective compounds with appropriate rodent pharmacokinetic properties has hampered the assessment of the effects of Cat C inhibitors on the activation of disease-relevant proteases in vivo and the full evaluation of the therapeutic utility of Cat C inhibitors.

Antiretroviral resistance at virological failure in the NEAT 001/ANRS 143 trial: raltegravir plus darunavir/ritonavir or tenofovir/emtricitabine plus darunavir/ritonavir as first-line ART.

PubMed

Lambert-Niclot, S; George, E C; Pozniak, A; White, E; Schwimmer, C; Jessen, H; Johnson, M; Dunn, D; Perno, C F; Clotet, B; Plettenberg, A; Blaxhult, A; Palmisano, L; Wittkop, L; Calvez, V; Marcelin, A G; Raffi, F

2016-04-01

To describe the pattern of drug resistance at virological failure in the NEAT001/ANRS143 trial (first-line treatment with ritonavir-boosted darunavir plus either tenofovir/emtricitabine or raltegravir). Genotypic testing was performed at baseline for reverse transcriptase (RT) and protease genes and for RT, protease and integrase (IN) genes for patients with a confirmed viral load (VL) >50 copies/mL or any single VL >500 copies/mL during or after week 32. A resistance test was obtained for 110/805 (13.7%) randomized participants qualifying for resistance analysis (61/401 of participants in the raltegravir arm and 49/404 of participants in the tenofovir/emtricitabine arm). No resistance-associated mutation (RAM) was observed in the tenofovir/emtricitabine plus darunavir/ritonavir arm, and all further analyses were limited to the raltegravir plus darunavir arm. In this group, 15/55 (27.3%) participants had viruses with IN RAMs (12 N155H alone, 1 N155H + Q148R, 1 F121Y and 1 Y143C), 2/53 (3.8%) with nucleotide analogue RT inhibitor RAMs (K65R, M41L) and 1/57 (1.8%) with primary protease RAM (L76V). The frequency of IN mutations at failure was significantly associated with baseline VL: 7.1% for a VL of <100,000 copies/mL, 25.0% for a VL of ≥100,000 copies/mL and <500,000 copies/mL and 53.8% for a VL of ≥500,000 copies/mL (PTREND = 0.007). Of note, 4/15 participants with IN RAM had a VL < 200 copies/mL at time of testing. In the NEAT001/ANRS143 trial, there was no RAM at virological failure in the standard tenofovir/emtricitabine plus darunavir/ritonavir regimen, contrasting with a rate of 29.5% (mostly IN mutations) in the raltegravir plus darunavir/ritonavir NRTI-sparing regimen. The cumulative risk of IN RAM after 96 weeks of follow-up in participants initiating ART with raltegravir plus darunavir/ritonavir was 3.9%. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

PubMed

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

2017-01-01

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

PubMed

Fang, Evandro Fei; Ng, Tzi Bun

2015-04-01

Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

Structural investigation of HIV-1 nonnucleoside reverse transcriptase inhibitors: 2-Aryl-substituted benzimidazoles

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-11-01

Acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most destructive epidemics in history. Inhibitors of HIV enzymes are the main targets to develop drugs against that disease. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic. Structural studies provide information necessary to design more active compounds. The crystal structures of four NNRTI derivatives of 2-aryl-substituted N-benzyl-benzimidazole are presented here. Analysis of the geometrical parameters shows that the structures of the investigated inhibitors are rigid. The important geometrical parameter is the dihedral angle between the planes of the π-electron systems of the benzymidazole and benzyl moieties. The values of these dihedral angles are in a narrow range for all investigated inhibitors. There is no significant difference between the structure of the free inhibitor and the inhibitor in the complex with RT HIV-1. X-ray structures of the investigated inhibitors are a good basis for modeling enzyme-inhibitor interactions in rational drug design.

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

PubMed

Rodriguez Orengo, J F; Santana, J; Febo, I; Diaz, C; Rodriguez, J L; Garcia, R; Font, E; Rosario, O

2000-03-01

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

Molecular Epidemiology of Norovirus Outbreaks in Norway during 2000 to 2005 and Comparison of Four Norovirus Real-Time Reverse Transcriptase PCR Assays

PubMed Central

Vainio, Kirsti; Myrmel, Mette

2006-01-01

During the period from January 2000 to August 2005 a total of 204 outbreaks of norovirus gastroenteritis were diagnosed at the Norwegian Institute of Public Health. A clear increase in the norovirus activity was seen in healthcare institutions during the winter seasons. Polymerase sequence analysis of norovirus strains from 122 outbreaks showed that 112 were caused by GII strains (91.8%). Two norovirus variants seen during the study period—GIIb and GII.4—were predominant between January 2000 and September 2002, whereas GII.4 was predominant from September 2002 onward. The highest norovirus activity was seen during the 2002-2003 and 2004-2005 seasons with the emergence of new GII.4 variants. This study describes the molecular epidemiology of norovirus strains circulating in Norway during the five previous seasons and compares four norovirus real-time reverse transcriptase PCR assays. A suitable assay for routine diagnostics is suggested. PMID:17021099

Gene 2 of the sigma rhabdovirus genome encodes the P protein, and gene 3 encodes a protein related to the reverse transcriptase of retroelements.

PubMed

Landès-Devauchelle, C; Bras, F; Dezélée, S; Teninges, D

1995-11-10

The nucleotide sequence of the genes 2 and 3 of the Drosophila rhabdovirus sigma was determined from cDNAs to viral genome and poly(A)+ mRNAs. Gene 2 comprises 1032 nucleotides and contains a long ORF encoding a molecular weight 35,208 polypeptide present in infected cells and in virions which migrates in SDS-PAGE as a doublet of M(r) about 60 kDa. The distribution of acidic charges as well as the electrophoretic properties of the protein are characteristic of the rhabdovirus P proteins. Gene 3 comprises 923 nucleotides and contains a long ORF capable of coding a polypeptide of 298 amino acids of MW 33,790. The putative protein (PP3) is similar in size to a minor component of the virions. Computer analysis shows that the sequence of PP3 contains three motifs related to the conserved motifs of reverse transcriptases.

TOPICAL TENOFOVIR, A MICROBICIDE EFFECTIVE AGAINST HIV, INHIBITS HERPES SIMPLEX VIRUS-2 REPLICATION

PubMed Central

Andrei, Graciela; Lisco, Andrea; Vanpouille, Christophe; Introini, Andrea; Balestra, Emanuela; van den Oord, Joost; Cihlar, Tomas; Perno, Carlo-Federico; Snoeck, Robert; Margolis, Leonid; Balzarini, Jan

2011-01-01

SUMMARY The HIV reverse transcriptase inhibitor tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and surprisingly herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct anti-herpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D-rafts, decreases HSV replication in human lymphoid and cervical tissues ex vivo, and delays HSV-induced lesions and death of topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse transcriptase. Tenofovir must be topically administered to achieve concentrations, which are higher than systemic levels after oral treatment, that exert these dual antiviral effects. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its co-pathogens. PMID:22018238

Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

PubMed Central

Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

2016-01-01

Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage. PMID:26613339

Expression and characterization of a novel reverse transcriptase of the LTR retrotransposon Tf1.

PubMed

Kirshenboim, Noa; Hayouka, Zvi; Friedler, Assaf; Hizi, Amnon

2007-09-30

The LTR retrotransposon of Schizosacharomyces pombe, Tf1, has several distinctive properties that can be related to the unique properties of its reverse transcriptase (RT). Consequently, we expressed, purified and studied the recombinant Tf1 RT. This monomeric protein possesses all activities typical to RTs: DNA and RNA-dependent DNA polymerase as well as an inherent ribonuclease H. The DNA polymerase activity shows preference to Mn(+)(2) or Mg(+)(2), depending on the substrate used, whereas the ribonuclease H strongly prefers Mn(+)(2). The most outstanding feature of Tf1 RT is its capacity to add non-templated nucleotides to the 3'-ends of the nascent DNA. This is mainly apparent in the presence of Mn(+)(2), as is the noticeable low fidelity of DNA synthesis. In all, Tf1 RT has a marked infidelity in synthesizing DNA at template ends, a phenomenon that can explain, as discussed herein, some of the features of Tf1 replication in the host cells.

The future of pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection.

PubMed

Özdener, Ayşe Elif; Park, Tae Eun; Kalabalik, Julie; Gupta, Rachna

2017-05-01

People at high risk for HIV acquisition should be offered pre-exposure prophylaxis (PrEP). Tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) is currently the only medication recommended for pre-exposure prophylaxis (PrEP) by the Centers for Disease Control and Prevention (CDC) in people at high risk for HIV acquisition. This article will review medications currently under investigation and the future landscape of PrEP therapy. Areas covered: This article will review clinical trials that have investigated nontraditional regimens of TDF/FTC, antiretroviral agents from different drug classes such as integrase strand transfer inhibitors (INSTI), nucleoside reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) as potential PrEP therapies. Expert commentary: Currently, there are several investigational drugs in the pipeline for PrEP against HIV infection. Increased utilization of PrEP therapy depends on provider identification of people at high risk for HIV transmission. Advances in PrEP development will expand options and access for people and reduce the risk of HIV acquisition.

Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

NASA Astrophysics Data System (ADS)

Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

2013-03-01

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase (RT), which is an essential enzyme for the replication of HIV. These changes allow it to retain potency against mutations that otherwise would render the enzyme resistant. Here we report that water molecules play an essential role in this binding process. Femtosecond experiments and theory expose the molecular level dynamics of rilpivirine bound to HIV-1 RT. Two nitrile substituents, one on each arm of the drug, are used as vibrational probes of the structural dynamics within the binding pocket. Two-dimensional vibrational echo spectroscopy reveals that one nitrile group is unexpectedly hydrogen-bonded to a mobile water molecule, not identified in previous X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure also reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond may help retain the potency of rilpivirine against pocket mutations despite the structural variations they cause.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei; Marx, Ailie

2017-01-01

Abstract Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors. PMID:28973474

Reverse transcriptase inhibitors as microbicides.

PubMed

Lewi, Paul; Heeres, Jan; Ariën, Kevin; Venkatraj, Muthusamy; Joossens, Jurgen; Van der Veken, Pieter; Augustyns, Koen; Vanham, Guido

2012-01-01

The CAPRISA 004 study in South Africa has accelerated the development of vaginal and rectal microbicides containing antiretrovirals that target specific enzymes in the reproduction cycle of HIV, especially reverse transcriptase inhibitors (RTI). In this review we discuss the potential relevance of HIV-1 RTIs as microbicides, focusing in the nucleotide RTI tenofovir and six classes of nonnucleoside RTIs (including dapivirine, UC781, urea and thiourea PETTs, DABOs and a pyrimidinedione). Although tenofovir and dapivirine appear to be most advanced in clinical trials as potential microbicides, several issues remain unresolved, e.g., the importance of nonhuman primates as a "gatekeeper" for clinical trials, the emergence and spread of drug-resistant mutants, the combination of microbicides that target different phases of viral reproduction and the accessibility to microbicides in low-income countries. Thus, here we discuss the latest research on RTI as microbicides in the light of the continuing spread of the HIV pandemic from the point of view of medicinal chemistry, virological, and pharmaceutical studies.

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

PubMed

Corbett, J W; Ko, S S; Rodgers, J D; Jeffrey, S; Bacheler, L T; Klabe, R M; Diamond, S; Lai, C M; Rabel, S R; Saye, J A; Adams, S P; Trainor, G L; Anderson, P S; Erickson-Viitanen, S K

1999-12-01

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Dual HIV-1 reverse transcriptase and integrase inhibitors from Limonium morisianum Arrigoni, an endemic species of Sardinia (Italy).

PubMed

Sanna, Cinzia; Rigano, Daniela; Corona, Angela; Piano, Dario; Formisano, Carmen; Farci, Domenica; Franzini, Genni; Ballero, Mauro; Chianese, Giuseppina; Tramontano, Enzo; Taglialatela-Scafati, Orazio; Esposito, Francesca

2018-02-04

During our search for potential templates of HIV-1 reverse transcriptase (RT) and integrase (IN) dual inhibitors, the methanolic extract obtained from aerial parts of Limonium morisianum was investigated. Repeated bioassay-guided chromatographic purifications led to the isolation of the following secondary metabolites: myricetin, myricetin 3-O-rutinoside, myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside, (-)-epigallocatechin 3-O-gallate, tryptamine, ferulic and phloretic acids. The isolated compounds were tested on both HIV-1 RT-associated RNase H and IN activities. Interestingly, (-)-epigallocatechin-3-O-gallate and myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside potently inhibited both enzyme activities with IC 50 values ranging from 0.21 to 10.9 μM. Differently, tryptamine and ferulic acid exhibited a significant inhibition only on the IN strand transfer reaction, showing a selectivity for this viral enzyme. Taken together these results strongly support the potential of this plant as a valuable anti HIV-1 drugs source worthy of further investigations.

The ORF1 Protein Encoded by LINE-1: Structure and Function During L1 Retrotransposition

PubMed Central

Martin, Sandra L.

2006-01-01

LINE-1, or L1 is an autonomous non-LTR retrotransposon in mammals. Retrotransposition requires the function of the two, L1-encoded polypeptides, ORF1p and ORF2p. Early recognition of regions of homology between the predicted amino acid sequence of ORF2 and known endonuclease and reverse transcriptase enzymes led to testable hypotheses regarding the function of ORF2p in retrotransposition. As predicted, ORF2p has been demonstrated to have both endonuclease and reverse transcriptase activities. In contrast, no homologs of known function have contributed to our understanding of the function of ORF1p during retrotransposition. Nevertheless, significant advances have been made such that we now know that ORF1p is a high affinity RNA binding protein that forms a ribonucleoprotein particle together with L1 RNA. Furthermore, ORF1p is a nucleic acid chaperone and this nucleic acid chaperone activity is required for L1 retrotransposition. PMID:16877816

Inhibitory effect of totarol on exotoxin proteins hemolysin and enterotoxins secreted by Staphylococcus aureus.

PubMed

Shi, Ce; Zhao, Xingchen; Li, Wenli; Meng, Rizeng; Liu, Zonghui; Liu, Mingyuan; Guo, Na; Yu, Lu

2015-10-01

Staphylococcus aureus (S. aureus) causes a wide variety of infections, which are of major concern worldwide. S. aureus produces multiple virulence factors, resulting in food infection and poisoning. These virulence factors include hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases and enterotoxins. Among the extracellular proteins produced by S. aureus that contribute to pathogenicity, the exotoxins α-hemolysin, staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) are thought to be of major significance. Totarol, a plant extract, has been revealed to inhibit the proliferation of several pathogens effectively. However, there are no reports on the effects of totarol on the production of α-hemolysin, SEA or SEB secreted by S. aureus. The aim of this study was to evaluate the effects of totarol on these three exotoxins. Hemolysis assay, western blotting and real-time reverse transcriptase-PCR assay were performed to identify the influence of graded subinhibitory concentrations of totarol on the production of α-hemolysin and the two major enterotoxins, SEA and SEB, by S. aureus in a dose-dependent manner. Moreover, an enzyme linked immunosorbent assay showed that the TNF-α production of RAW264.7 cells stimulated by S. aureus supernatants was inhibited by subinhibitory concentrations of totarol. Form the data, we propose that totarol could potentially be used as a promising natural compound in the food and pharmaceutical industries.

PpRT1: the first complete gypsy-like retrotransposon isolated in Pinus pinaster.

PubMed

Rocheta, Margarida; Cordeiro, Jorge; Oliveira, M; Miguel, Célia

2007-02-01

We have isolated and characterized a complete retrotransposon sequence, named PpRT1, from the genome of Pinus pinaster. PpRT1 is 5,966 bp long and is closely related to IFG7 gypsy retrotransposon from Pinus radiata. The long terminal repeats (LTRs) have 333 bp each and show a 5.4% sequence divergence between them. In addition to the characteristic polypurine tract (PPT) and the primer binding site (PBS), PpRT1 carries internal regions with homology to retroviral genes gag and pol. The pol region contains sequence motifs related to the enzymes protease, reverse transcriptase, RNAseH and integrase in the same typical order known for Ty3/gypsy-like retrotransposons. PpRT1 was extended from an EST database sequence indicating that its transcription is occurring in pine tissues. Southern blot analyses indicate however, that PpRT1 is present in a unique or a low number of copies in the P. pinaster genome. The differences in nucleotide sequence found between PpRT1 and IFG7 may explain the strikingly different copy number in the two pine species genome. Based on the homologies observed when comparing LTR region among different gypsy elements we propose that the highly conserved LTR regions may be useful to amplify other retrotransposon sequences of the same or close retrotransposon family.

Update on HIV resistance and resistance testing.

PubMed

Sebastian, Joseph; Faruki, Hawazin

2004-01-01

The introduction of highly active antiretroviral therapy, including a combination of antivirals directed at various steps in the viral life cycle, has led to significant decreases in morbidity and mortality associated with human immunodeficiency virus (HIV-1) infections. Despite the availability of numerous antivirals, many extensively treated patients gradually loose the ability to control viral replication because of development of antiviral resistance. Laboratory tests have been developed and validated to assist in recognizing such resistance and to help predict which antivirals may be more likely to control viral replication in a given patient. Both genotypic and phenotypic assays have been developed to assess HIV-1 antiviral resistance. The assay methodologies, including the advantages and disadvantages of each method, as well as the limitations of each method are reviewed. The ability to predict likely drug response from a genotype or a phenotype is continually evolving, and the more recently discovered mutation/drug resistance associations are discussed in terms of their implications for HIV resistance assays. To provide additional options for those who have developed resistance to all currently available drugs, new antivirals, such as the fusion inhibitors, are being developed. These new classes of antivirals block the HIV viral life cycle at sites other than reverse transcriptase and protease. Unique and novel resistance assays are being developed to measure HIV resistance to these new drugs. Copyright 2003 Wiley Periodicals, Inc.

Mathematical analysis of antiretroviral therapy aimed at HIV-1 eradication or maintenance of low viral loads.

PubMed

Wein, L M; D'Amato, R M; Perelson, A S

1998-05-07

Motivated by the ability of combinations of antiretroviral agents to sustain viral suppression in HIV-1-infected individuals, we analyse the transient and steady-state behavior of a mathematical model of HIV-1 dynamics in vivo in order to predict whether these drug regimens can eradicate HIV-1 or maintain viral loads at low levels. The model incorporates two cell types (CD4+ T cells and a long-lived pool of cells), two strains of virus (drug-sensitive wild type and drug-resistant mutant) and two types of antiretroviral agents (reverse transcriptase and protease inhibitors). The transient behavior of the cells and virus and the eventual eradication of the virus are determined primarily by the strength of the combination therapy against the mutant strain and the maximum achievable increase in the uninfected CD4+ T cell concentration. We also predict, if the parameters of the model remain constant during therapy, that less intensive maintenance regimens will be unable to maintain low viral loads for extensive periods of time. However, if the reduction in viral load produced by therapy reduces the state of activation of the immune system, the number of cells susceptible for HIV-1 infection may decrease even though total CD4+ T cells increase. Our model predicts that if this occurs strong inductive therapy that reduces viral load followed by weaker maintenance regimes may succeed.

HIVprotI: an integrated web based platform for prediction and design of HIV proteins inhibitors.

PubMed

Qureshi, Abid; Rajput, Akanksha; Kaur, Gazaldeep; Kumar, Manoj

2018-03-09

A number of anti-retroviral drugs are being used for treating Human Immunodeficiency Virus (HIV) infection. Due to emergence of drug resistant strains, there is a constant quest to discover more effective anti-HIV compounds. In this endeavor, computational tools have proven useful in accelerating drug discovery. Although methods were published to design a class of compounds against a specific HIV protein, but an integrated web server for the same is lacking. Therefore, we have developed support vector machine based regression models using experimentally validated data from ChEMBL repository. Quantitative structure activity relationship based features were selected for predicting inhibition activity of a compound against HIV proteins namely protease (PR), reverse transcriptase (RT) and integrase (IN). The models presented a maximum Pearson correlation coefficient of 0.78, 0.76, 0.74 and 0.76, 0.68, 0.72 during tenfold cross-validation on IC 50 and percent inhibition datasets of PR, RT, IN respectively. These models performed equally well on the independent datasets. Chemical space mapping, applicability domain analyses and other statistical tests further support robustness of the predictive models. Currently, we have identified a number of chemical descriptors that are imperative in predicting the compound inhibition potential. HIVprotI platform ( http://bioinfo.imtech.res.in/manojk/hivproti ) would be useful in virtual screening of inhibitors as well as designing of new molecules against the important HIV proteins for therapeutics development.

Impact of Noncoding Satellite Repeats on Pancreatic Cancer Metastasis

DTIC Science & Technology

2014-09-01

nucleoside reverse transcriptase inhibitor ddC as a small molecule inhibitor of HSATII reverse transcription. Initial data indicates there are anti...proliferative effects of ddC in cancer cell lines. We will evaluate ddC and anti-sense locked nucleic acids as methods for inhibiting this process and...of these hybrids, we tested the effect of the nucleoside analog RT inhibitor (NRTI) 2’,3’-dideoxycytidine ( ddC ) in COLO205 cells (Fig. 2e). Notably

The CD4/CD8 Ratio is Inversely Associated with Carotid Intima-Media Thickness Progression in Human Immunodeficiency Virus-Infected Patients on Antiretroviral Treatment.

PubMed

Bernal Morell, Enrique; Serrano Cabeza, José; Muñoz, Ángeles; Marín, Irene; Masiá, Mar; Gutiérrez, Félix; Cano, Alfredo

2016-07-01

Inversion of the CD4/CD8 ratio (<1) has been identified as a surrogate marker of immunosenescence and an independent predictor of AIDS events in HIV-infected patients and mortality in the general population. We aimed to assess the association between the CD4/CD8 ratio and carotid intima-media thickness (cIMT) progression in treated HIV-infected patients as a marker of coronary heart disease. A longitudinal study was conducted during 3 years in 96 virally suppressed HIV-infected patients receiving antiretroviral treatment (ART). We analyzed the associations between the CD4/CD8 ratio, cardiovascular risk factors, and antiretroviral treatment (ART) and progression of subclinical atherosclerosis assessed using cIMT at baseline and after 3 years. Finally, 96 patients completed the study. Seventy six (79.1%) patients were male, aged 44 ± 10 years; 39 (40.6%) were on treatment with protease inhibitors; 49 (51.04%) with nonnucleoside reverse transcriptase inhibitors, 6 (6.25%) with integrase inhibitors, 3 (3.12%) with maraviroc, and 2 (2.08%) just with nucleoside reverse transcriptase inhibitors. The mean of ART exposition was 6.9 ± 5.9 years. Twenty six (27%) patients had family history of ischemic heart disease, 51 (53.12%) were smokers, 12 (12.5%) were hypertensive, 4 (4.16%) had type 2 diabetes, 23 (23.9%) had dyslipidemia, and 31 (32.3%) were infected with hepatitis C virus. Baseline cIMT was significantly associated with age (rho = 0.497; p < .001), basal glucemia (rho = 0.323; p = .001), triglycerides (rho = 0.232; p = .023), Framingham risk score (rho = 0.324; p = .001), CD4/CD8 ratio (rho = -0.176; p = .05), and dyslipidemia (0.72 ± 0.16 mm vs. 0.63 ± 0.11 mm; p = .029). In multivariable analysis where cardiovascular risk factor and ART were included, cIMT progression was inversely associated with CD4/CD8 ratio [odds ratio (OR) = 0.283; confidence interval (95% CI) 0.099-0.809; p = .019]. In conclusion, the inversion of CD4/CD8 ratio in treated HIV-infected patients is independently associated with cIMT progression, a marker of coronary heart disease. Therefore, it might be clinically useful as predictor of cardiovascular events.

[GeSIDA/National AIDS Plan: Consensus document on antiretroviral therapy in adults infected by the human immunodeficiency virus (Updated January 2014)].

PubMed

2014-01-01

This consensus document is an update of combined antiretroviral therapy (cART) guidelines for HIV-1 infected adult patients. To formulate these recommendations a panel composed of members of the Grupo de Estudio de Sida and the Plan Nacional sobre el Sida reviewed the efficacy and safety advances in clinical trials, cohort and pharmacokinetic studies published in medical journals (PubMed and Embase) or presented in medical scientific meetings. Recommendations strength and the evidence in which they are supported are based on modified criteria of the Infectious Diseases Society of America. In this update, antiretroviral therapy (ART) is recommended for all patients infected by type 1 human immunodeficiency virus (HIV-1). The strength and grade of the recommendation varies with the clinical circumstances: CDC stage B or C disease (A-I), asymptomatic patients (depending on the CD4+ T-lymphocyte count: <350cells/μL, A-I; 350-500 cells/μL, A-II, and >500 cells/μL, B-III), comorbid conditions (HIV nephropathy, chronic hepatitis caused by HBV or HCV, age >55years, high cardiovascular risk, neurocognitive disorders, and cancer, A-II), and prevention of transmission of HIV (mother-to-child or heterosexual, A-I; men who have sex with men, A-III). The objective of ART is to achieve an undetectable plasma viral load. Initial ART should always comprise a combination of 3 drugs, including 2 nucleoside reverse transcriptase inhibitors and a third drug from a different family (non-nucleoside reverse transcriptase inhibitor, protease inhibitor, or integrase inhibitor). Some of the possible initial regimens have been considered alternatives. This update presents the causes and criteria for switching ART in patients with undetectable plasma viral load and in cases of virological failure where rescue ART should comprise 2 or 3 drugs that are fully active against the virus. An update is also provided for the specific criteria for ART in special situations (acute infection, HIV-2 infection, and pregnancy) and with comorbid conditions (tuberculosis or other opportunistic infections, kidney disease, liver disease, and cancer). These new guidelines updates previous recommendations related to cART (when to begin and what drugs should be used), how to monitor and what to do in case of viral failure or drug adverse reactions. cART specific criteria in comorbid patients and special situations are equally updated. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

API consensus guidelines for use of antiretroviral therapy in adults (API-ART guidelines). Endorsed by the AIDS Society of India.

PubMed

Gupta, S B; Pujari, S N; Joshi, S R; Patel, A K

2006-01-01

With rational use of antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection has been transformed into a chronic manageable illness like diabetes and hypertension. These guidelines provide information on state of art, evidence based approach for use of ART in Indian context. When to initiate ART? Antiretroviral therapy is indicated for all symptomatic HIV infected persons regardless of CD4 counts and plasma viral load (PVL) levels. In asymptomatic patients, ART should be offered when the CD4 counts < 200/mm3 and should be considered in patients with CD4 counts between 200-250/mm3. Therapy is not recommended for patients with CD4 count more than 350/ mm3. Involvement of patient in all treatment decisions and assessing readiness is critical before initiating ART. What to start with? A non-nucleoside reverse transcriptase inhibitor (NNRTI) based regimen is recommended for antiretroviral naïve patients. The choice between nevirapine and efavirenz is based on differences in adverse events profiles; cost and availability of convenient fixed dose combinations and need for concomitant use of rifampicin. A backbone of 2-nucleoside reverse transcriptase inhibitors (NRTIs) is combined with the NNRTI. Various combinations and ART strategies not to be used in clinical practice has been enlisted. How to follow up? Recommendations have been made for baseline evaluation and monitoring of patients on ART. These include guidelines on laboratory and clinical evaluation. A plasma viral load at 6 months after initiation of first-line ART is strongly recommended. Yearly estimation of lipid profile has been recommended. How to identify and manage ART failure? The guidelines recognize the issue of identifying ART failure late if only CD4 counts are used for monitoring. In the absence of resistance testing various second-line regimens have been enlisted. A boosted protease inhibitor based regimen is recommended in this situation to be combined with 2-NRTIs. Special situations Recommendations have been made for use of ART in HIV-TB, HIV-HBV, and HIV-HCV co-infected patients. In patients with active TB and a CD4 count < 200/mm3, initiation of ART is recommended as soon as the anti-TB treatment is tolerated. Efavirenz is the only ARV drug, which can be safely used with rifampicin. In pregnancy use of single dose nevirapine for reducing risk of mother to child transmission of HIV is not recommended, because of the risk of development of resistance. For post-exposure prophylaxis taking ART treatment history of the source patient is crucial in designing an effective regimen.

Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2016 Recommendations of the International Antiviral Society-USA Panel.

PubMed

Günthard, Huldrych F; Saag, Michael S; Benson, Constance A; del Rio, Carlos; Eron, Joseph J; Gallant, Joel E; Hoy, Jennifer F; Mugavero, Michael J; Sax, Paul E; Thompson, Melanie A; Gandhi, Rajesh T; Landovitz, Raphael J; Smith, Davey M; Jacobsen, Donna M; Volberding, Paul A

2016-07-12

New data and therapeutic options warrant updated recommendations for the use of antiretroviral drugs (ARVs) to treat or to prevent HIV infection in adults. To provide updated recommendations for the use of antiretroviral therapy in adults (aged ≥18 years) with established HIV infection, including when to start treatment, initial regimens, and changing regimens, along with recommendations for using ARVs for preventing HIV among those at risk, including preexposure and postexposure prophylaxis. A panel of experts in HIV research and patient care convened by the International Antiviral Society-USA reviewed data published in peer-reviewed journals, presented by regulatory agencies, or presented as conference abstracts at peer-reviewed scientific conferences since the 2014 report, for new data or evidence that would change previous recommendations or their ratings. Comprehensive literature searches were conducted in the PubMed and EMBASE databases through April 2016. Recommendations were by consensus, and each recommendation was rated by strength and quality of the evidence. Newer data support the widely accepted recommendation that antiretroviral therapy should be started in all individuals with HIV infection with detectable viremia regardless of CD4 cell count. Recommended optimal initial regimens for most patients are 2 nucleoside reverse transcriptase inhibitors (NRTIs) plus an integrase strand transfer inhibitor (InSTI). Other effective regimens include nonnucleoside reverse transcriptase inhibitors or boosted protease inhibitors with 2 NRTIs. Recommendations for special populations and in the settings of opportunistic infections and concomitant conditions are provided. Reasons for switching therapy include convenience, tolerability, simplification, anticipation of potential new drug interactions, pregnancy or plans for pregnancy, elimination of food restrictions, virologic failure, or drug toxicities. Laboratory assessments are recommended before treatment, and monitoring during treatment is recommended to assess response, adverse effects, and adherence. Approaches are recommended to improve linkage to and retention in care are provided. Daily tenofovir disoproxil fumarate/emtricitabine is recommended for use as preexposure prophylaxis to prevent HIV infection in persons at high risk. When indicated, postexposure prophylaxis should be started as soon as possible after exposure. Antiretroviral agents remain the cornerstone of HIV treatment and prevention. All HIV-infected individuals with detectable plasma virus should receive treatment with recommended initial regimens consisting of an InSTI plus 2 NRTIs. Preexposure prophylaxis should be considered as part of an HIV prevention strategy for at-risk individuals. When used effectively, currently available ARVs can sustain HIV suppression and can prevent new HIV infection. With these treatment regimens, survival rates among HIV-infected adults who are retained in care can approach those of uninfected adults.

Suicide Inhibitors of Reverse Transcriptase in the Therapy of AIDS and Other Retroviruses

DTIC Science & Technology

1990-07-01

I and 10 nanonolar) and compared to the E . Coli recombinant HIV-RT (Kindly donated by Dr. Steven Hughes Fort Detrick M.D.) and the wild type HIV-RT...Both the wild type and E . Coli HIV-RT’s were resistant to PFA showing essentially no inhibition at the lOnM level. Previous studies have shown that...10 nanomolar PFA. j, Sentivitv of Recombinant HIV-Reverse Transcriotase to Foscarnet. RECOMBINANT HIV RT ( E . COLI) + FOSCARNET 350001R 300000 PFA .001

Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

DTIC Science & Technology

2007-05-08

deoxynucleotide triphosphates, from Sigma. Sequences for glyceraldehyde-3-phosphate dehydrogenase ( G3PDH ), IL-8,and TNF-a were amplified with primer...This was accomplished by normalizing all samples to the mRNA for the moderately expressed housekeeping function glyceraldehyde-3 -phosphate...without and with isolation of cells before reverse transcription and PCR. G3PDH mRNA target amplifies at 983 base pairs. The 630 base pair band is the

First report of Cocksfoot mottle virus infecting wheat (Triticum aestivum) in Ohio

USDA-ARS?s Scientific Manuscript database

Cocksfoot mottle virus (CfMV) was discovered in Ohio wheat during a 2016 field survey utilizing RNA-Seq to identify virus-like sequences. Virus sequences were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing, and CfMV was transmitted to orchardgrass and pas...

RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts

EPA Science Inventory

The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion p...

Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2008-01-01

Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

Isolation and characterization of an AGAMOUS homolog from Fraxinus pennsylvanica

Treesearch

Ningxia Du; Paula M. Pijut

2010-01-01

An AGAMOUS homolog (FpAG) was isolated from green ash (Fraxinus pennsylvanica) using a reverse transcriptase polymerase chain reaction method. Southern blot analysis indicated that FpAG was present as a single-copy sequence in the genome of green ash. RNA accumulated in the reproductive tissues (female...

In-vitro Cell Culture and Real-time Reverse Transcriptase PCR-based Assays to Detect Infective Toxoplas gondii Oocysts

EPA Science Inventory

Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canad...

Quantification of HTLV-1 reverse transcriptase activity in ATL patients treated with zidovudine and interferon-α.

PubMed

Macchi, Beatrice; Balestrieri, Emanuela; Frezza, Caterina; Grelli, Sandro; Valletta, Elena; Marçais, Ambroise; Marino-Merlo, Francesca; Turpin, Jocelyn; Bangham, Charles R; Hermine, Olivier; Mastino, Antonio; Bazarbachi, Ali

2017-05-09

The therapeutic efficacy of the AZT and IFN combination in ATL presumably reflects the inhibition of RT-related functions.HTLV-1-RT activity from short-term cultured PBMCs may represent a predictive correlate of clinical response to AZT/IFN in ATL patients.

Serine protease activity in m-1 cortical collecting duct cells.

PubMed

Liu, Lian; Hering-Smith, Kathleen S; Schiro, Faith R; Hamm, L Lee

2002-04-01

An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription-polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I(eq)), which represents sodium transport in these cells, dropped to 59+/-3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I(eq) in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I(eq) but also reduced transepithelial resistance (R(te)) to 43+/-2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R(te). Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I(eq) in M-1 cells. STI inhibited I(eq) gradually over 6 hours, and the inhibition of I(eq) by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity or prostasin mRNA levels, and in fact, dexamethasone decreased prostasin mRNA expression. In conclusion, although prostasin is present in M-1 cells and probably augments sodium transport in these cells, serine proteases probably have other effects (eg, resistance) in the collecting duct in addition to effects on sodium channels. Steroids do not alter these effects in M-1 cells. Additional proteases are likely also present in mouse collecting duct cells.

Identification and characterization of jute LTR retrotransposons:

PubMed Central

Ahmed, Salim; Shafiuddin, MD; Azam, Muhammad Shafiul; Islam, Md. Shahidul; Ghosh, Ajit

2011-01-01

Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome. PMID:22016842

Active methamphetamine use is associated with transmitted drug resistance to non-nucleoside reverse transcriptase inhibitors in individuals with HIV infection of unknown duration.

PubMed

Cachay, Edward R; Moini, Niousha; Kosakovsky Pond, Sergei L; Pesano, Rick; Lie, Yolanda S; Aiem, Heidi; Butler, David M; Letendre, Scott; Mathews, Wm Christopher; Smith, Davey M

2007-01-01

Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Cross-sectional analysis. Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI.

Naringin Reverses Hepatocyte Apoptosis and Oxidative Stress Associated with HIV-1 Nucleotide Reverse Transcriptase Inhibitors-Induced Metabolic Complications

PubMed Central

Adebiyi, Oluwafeyisetan O.; Adebiyi, Olubunmi A.; Owira, Peter M. O.

2015-01-01

Nucleoside Reverse Transcriptase Inhibitors (NRTIs) have not only improved therapeutic outcomes in the treatment of HIV infection but have also led to an increase in associated metabolic complications of NRTIs. Naringin’s effects in mitigating NRTI-induced complications were investigated in this study. Wistar rats, randomly allotted into seven groups (n = 7) were orally treated daily for 56 days with 100 mg/kg zidovudine (AZT) (groups I, II III), 50 mg/kg stavudine (d4T) (groups IV, V, VI) and 3 mL/kg of distilled water (group VII). Additionally, rats in groups II and V were similarly treated with 50 mg/kg naringin, while groups III and VI were treated with 45 mg/kg vitamin E. AZT or d4T treatment significantly reduced body weight and plasma high density lipoprotein concentrations but increased liver weights, plasma triglycerides and total cholesterol compared to controls, respectively. Furthermore, AZT or d4T treatment significantly increased oxidative stress, adiposity index and expression of Bax protein, but reduced Bcl-2 protein expression compared to controls, respectively. However, either naringin or vitamin E significantly mitigated AZT- or d4T-induced weight loss, dyslipidemia, oxidative stress and hepatocyte apoptosis compared to AZT- or d4T-only treated rats. Our results suggest that naringin reverses metabolic complications associated with NRTIs by ameliorating oxidative stress and apoptosis. This implies that naringin supplements could mitigate lipodystrophy and dyslipidemia associated with NRTI therapy. PMID:26690471

Integration of maternal genome into the neonate genome through breast milk mRNA transcripts and reverse transcriptase.

PubMed

Irmak, M Kemal; Oztas, Yesim; Oztas, Emin

2012-06-07

Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain mRNA transcripts and possess reverse transcriptase activity. They contain about 14,000 transcripts representing the milk transcriptome. Microvesicles are also enriched with proteins related to "caveolar-mediated endocytosis signaling" pathway. It has recently been reported that microvesicles could be transferred to other cells by endocytosis and their RNA content can be translated and be functional in their new location. A significant percentage of the mammalian genome appears to be the product of reverse transcription, containing sequences whose characteristics point to RNA as a template precursor. These are mobile elements that move by way of transposition and are called retrotransposons. We thought that retrotransposons may stem from about 14,000 transcriptome of breast milk microvesicles, and reviewed the literature.The enhanced acceptance of maternal allografts in children who were breast-fed and tolerance to the maternal MHC antigens after breastfeeding may stem from RNAs of the breast milk microvesicles that can be taken up by the breastfed infant and receiving maternal genomic information. We conclude that milk microvesicles may transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Integration of maternal genome into the neonate genome through breast milk mRNA transcripts and reverse transcriptase

PubMed Central

2012-01-01

Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain mRNA transcripts and possess reverse transcriptase activity. They contain about 14,000 transcripts representing the milk transcriptome. Microvesicles are also enriched with proteins related to “caveolar-mediated endocytosis signaling” pathway. It has recently been reported that microvesicles could be transferred to other cells by endocytosis and their RNA content can be translated and be functional in their new location. A significant percentage of the mammalian genome appears to be the product of reverse transcription, containing sequences whose characteristics point to RNA as a template precursor. These are mobile elements that move by way of transposition and are called retrotransposons. We thought that retrotransposons may stem from about 14,000 transcriptome of breast milk microvesicles, and reviewed the literature. The enhanced acceptance of maternal allografts in children who were breast-fed and tolerance to the maternal MHC antigens after breastfeeding may stem from RNAs of the breast milk microvesicles that can be taken up by the breastfed infant and receiving maternal genomic information. We conclude that milk microvesicles may transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases. PMID:22676860

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

Overexpression of Telomerase Reverse Transcriptase Induces Autism-like Excitatory Phenotypes in Mice.

PubMed

Kim, Ki Chan; Rhee, Jeehae; Park, Jong-Eun; Lee, Dong-Keun; Choi, Chang Soon; Kim, Ji-Woon; Lee, Han-Woong; Song, Mi-Ryoung; Yoo, Hee Jeong; Chung, ChiHye; Shin, Chan Young

2016-12-01

In addition to its classical role as a regulator of telomere length, recent reports suggest that telomerase reverse transcriptase (TERT) plays a role in the transcriptional regulation of gene expression such as β-catenin-responsive pathways. Silencing or over-expression of TERT in cultured NPCs demonstrated that TERT induced glutamatergic neuronal differentiation. During embryonic brain development, expression of transcription factors involved in glutamatergic neuronal differentiation was increased in mice over-expressing TERT (TERT-tg mice). We observed increased expression of NMDA receptor subunits and phosphorylation of α-CaMKII in TERT-tg mice. TERT-tg mice showed autism spectrum disorder (ASD)-like behavioral phenotypes as well as lowered threshold against electrically induced seizure. Interestingly, the NMDA receptor antagonist memantine restored behavioral abnormalities in TERT-tg mice. Consistent with the alteration in excitatory/inhibitory (E/I) ratio, TERT-tg mice showed autism-like behaviors, abnormal synaptic organization, and function in mPFC suggesting the role of altered TERT activity in the manifestation of ASD, which is further supported by the significant association of certain SNPs in Korean ASD patients.

Synthesis, Biological Activity, and Crystal Structure of Potent Nonnucleoside Inhibitors of HIV-1 Reverse Transcriptase That Retain Activity against Mutant Forms of the Enzyme†

PubMed Central

Morningstar, Marshall L.; Roth, Thomas; Farnsworth, David W.; Smith, Marilyn Kroeger; Watson, Karen; Buckheit, Robert W.; Das, Kalyan; Zhang, Wanyi; Arnold, Eddy; Julias, John G.; Hughes, Stephen H.; Michejda, Christopher J.

2010-01-01

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 μM, EC50 = 0.44 μM, and TC50 ≥ 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants. PMID:17663538

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung

2008-01-05

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Reverse transcriptase polymerase chain reaction on fine needle aspirates for rapid detection of translocations in synovial sarcoma.

PubMed

Nilsson, G; Wang, M; Wejde, J; Kanter, L; Karlén, J; Tani, E; Kreicbergs, A; Larsson, O

1998-01-01

To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericytoma, one was a poorly defined tumor, and three were suspected synovial sarcomas. FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Crystal structures of the reverse transcriptase-associated ribonuclease H domain of xenotropic murine leukemia-virus related virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Dongwen; Chung, Suhman; Miller, Maria

2012-06-19

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site {alpha}-hydroxytropolone inhibitor. Enzymatic assays showed that the intactmore » RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with {beta}-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.« less

Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

PubMed

Miri-Moghaddam, Ebrahim; Deezagi, Abdolkhaleg; Soheili, Zahra Sohaila; Shariati, Parvin

2010-01-01

The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia. Copyright © 2010 S. Karger AG, Basel.

A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides.

PubMed Central

Smith, R A; Remington, K M; Lloyd, R M; Schinazi, R F; North, T W

1997-01-01

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides. PMID:9032372

A Laccase with HIV-1 Reverse Transcriptase Inhibitory Activity from the Broth of Mycelial Culture of the Mushroom Lentinus tigrinus

PubMed Central

Xu, LiJing; Wang, HeXiang; Ng, TziBun

2012-01-01

A 59 kDa laccase with inhibitory activity against HIV-1 reverse transcriptase (IC50 = 2.4 μM) was isolated from the broth of mycelial culture of the mushroom Lentinus tigrinus. The isolation procedure involved ion exchange chromatography on DEAE-cellulose and CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was adsorbed on both types of ion exchangers. About 95-fold purification was achieved with a 25.9% yield of the enzyme. The procedure resulted in a specific enzyme activity of 76.6 U/mg. Its N-terminal amino acid sequence was GIPDLHDLTV, which showed little similarity to other mushroom laccase and other Lentinus tigrinus strain laccase. Its characteristics were different from previously reported laccase of other Lentinus tigrinus strain. Maximal laccase activity was observed at a pH of 4 and at a temperature of 60°C, respectively. This study yielded the information about the potentially exploitable activities of Lentinus tigrinus laccase. PMID:22536022

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

PubMed

Toro, Nicolás; Martínez-Abarca, Francisco; González-Delgado, Alejandro

2017-08-02

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

PubMed

Urdea, M S; Wilber, J C; Yeghiazarian, T; Todd, J A; Kern, D G; Fong, S J; Besemer, D; Hoo, B; Sheridan, P J; Kokka, R

1993-11-01

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo

PubMed Central

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M.; Weissman, Jonathan S.; Rouskin, Silvi

2017-01-01

Coupling structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structural studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduces biases and necessitates population-average assessments of RNA structure. Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in non-canonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs to their mature isoforms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand in vivo analysis of RNA structure. PMID:27819661

Micronuclei induced by reverse transcriptase inhibitors in mononucleated and binucleated cells as assessed by the cytokinesis-block micronucleus assay

PubMed Central

2010-01-01

This study evaluated the clastogenic and/or aneugenic potential of three nucleoside reverse transcriptase inhibitors (zidovudine - AZT, lamivudine - 3TC and stavudine - d4T) using the cytokinesis-block micronucleus (CBMN) assay in human lymphocyte cultures. All three inhibitors produced a positive response when tested in binucleated cells. The genotoxicity of AZT and 3TC was restricted to binucleated cells since there was no significant increase in the frequency of micronuclei in mononucleated cells. This finding indicated that AZT and 3TC caused chromosomal breakage and that their genotoxicity was related to a clastogenic action. In addition to the positive response observed with d4T in binucleated cells, this drug also increased the frequency of micronuclei in mononucleated cells, indicating clastogenic and aneugenic actions. Since the structural differences between AZT and 3TC and AZT and d4T involve the 3' position in the 2'-deoxyribonucleoside and in an unsaturated 2',3',dideoxyribose, respectively, we suggest that an unsaturated 2', 3', dideoxyribose is responsible for the clastogenic and aneugenic actions of d4T. PMID:21637587

Characterization of UC781-Tenofovir Combination Gel Products for HIV-1 Infection Prevention in an Ex Vivo Ectocervical Model

PubMed Central

Cost, Marilyn; Dezzutti, Charlene S.; Clark, Meredith R.; Friend, David R.; Akil, Ayman

2012-01-01

HIV continues to be a problem worldwide. Topical vaginal microbicides represent one option being evaluated to stop the spread of HIV. With drug candidates that have a specific action against HIV now being studied, it is important that, when appropriate and based on the mechanism of action, the drug permeates the tissue so that it can be delivered to specific targets which reside there. Novel formulations of the nucleotide reverse transcriptase inhibitor tenofovir (TFV) and the nonnucleoside reverse transcriptase inhibitor UC781 have been developed and evaluated here. Gels with three distinct rheological properties were prepared. The three gels released both UC781 and TFV under in vitro conditions at concentrations equal to or above the reported 50% effective concentrations (EC50s). The drug concentrations in ectocervical tissues were well in excess of the reported EC50s. The gels maintain ectocervical viability and prevent infection of ectocervical explants after a HIV-1 challenge. This study successfully demonstrates the feasibility of using this novel combination of antiretroviral agents in an aqueous gel as an HIV infection preventative. PMID:22430977

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Efficacy and safety of TMC278 in antiretroviral-naive HIV-1 patients: week 96 results of a phase IIb randomized trial.

PubMed

Pozniak, Anton L; Morales-Ramirez, Javier; Katabira, Elly; Steyn, Dewald; Lupo, Sergio H; Santoscoy, Mario; Grinsztejn, Beatriz; Ruxrungtham, Kiat; Rimsky, Laurence T; Vanveggel, Simon; Boven, Katia

2010-01-02

TMC278 is a next-generation nonnucleoside reverse transcriptase inhibitor highly active against wild-type and nonnucleoside reverse transcriptase inhibitor-resistant HIV-1 in vitro. The week 96 analysis of TMC278-C204, a large dose-ranging study of TMC278 in treatment-naive HIV-1-infected patients, is presented. Phase IIb randomized trial. Three hundred sixty-eight patients were randomized and treated with three blinded once-daily TMC278 doses 25, 75 or 150 mg, or an open-label, active control, efavirenz 600 mg once daily, all with two nucleoside reverse transcriptase inhibitors. The primary analysis was at week 48. No TMC278 dose-response relationship for efficacy and safety was observed. TMC278 demonstrated potent antiviral efficacy comparable with efavirenz over 48 weeks that was sustained to week 96 (76.9-80.0% and 71.4-76.3% of TMC278-treated patients with confirmed viral load <50 copies/ml, respectively; time-to-loss of virological-response algorithm). Median increases from baseline in CD4 cell count with TMC278 at week 96 (138.0-149.0 cells/microl) were higher than at week 48 (108.0-123.0 cells/microl). All TMC278 doses were well tolerated. The incidences of the most commonly reported grade 2-4 adverse events at least possibly related to study medication, including nausea, dizziness, abnormal dreams/nightmare, dyspepsia, asthenia, rash, somnolence and vertigo, were low and lower with TMC278 than with efavirenz. Incidences of serious adverse events, grade 3 or 4 adverse events and discontinuations due to adverse events were similar among groups. All TMC278 doses demonstrated potent and sustained efficacy comparable with efavirenz in treatment-naive patients over 96 weeks. TMC278 was well tolerated with lower incidences of neurological and psychiatric adverse events, rash and lower lipid elevations than those with efavirenz. TMC278 25 mg once daily was selected for further clinical development.

Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

PubMed

Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

2016-05-01

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

A Combination Microbicide Gel Protects Macaques Against Vaginal Simian Human Immunodeficiency Virus-Reverse Transcriptase Infection, But Only Partially Reduces Herpes Simplex Virus-2 Infection After a Single High-Dose Cochallenge

PubMed Central

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D.; Fernández-Romero, Jose A.; Zydowsky, Thomas M.

2014-01-01

Abstract Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides. PMID:24117013

A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge.

PubMed

Hsu, Mayla; Aravantinou, Meropi; Menon, Radhika; Seidor, Samantha; Goldman, Daniel; Kenney, Jessica; Derby, Nina; Gettie, Agegnehu; Blanchard, James; Piatak, Michael; Lifson, Jeffrey D; Fernández-Romero, Jose A; Zydowsky, Thomas M; Robbiani, Melissa

2014-02-01

Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.

The HEPT Analogue WPR-6 Is Active against a Broad Spectrum of Nonnucleoside Reverse Transcriptase Drug-Resistant HIV-1 Strains of Different Serotypes.

PubMed

Xu, Weisi; Zhao, Jianxiong; Sun, Jianping; Yin, Qianqian; Wang, Yan; Jiao, Yang; Liu, Junyi; Jiang, Shibo; Shao, Yiming; Wang, Xiaowei; Ma, Liying

2015-08-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of the highly active antiretroviral therapy (HAART) used to treat human immunodeficiency type 1 virus (HIV-1). However, because of the emergence of drug resistance and the adverse effects of current anti-HIV drugs, it is essential to develop novel NNRTIs with an excellent safety profile, improved activity against NNRTI-resistant viruses, and enhanced activity against clinical isolates of different subtypes. Here, we have identified 1-[(benzyloxy)methyl]-6-(3,5-dimethylbenzyl)-5-iodopyrimidine-2,4(1H,3H)-dione (WPR-6), a novel NNRTI with a 50% effective concentration (EC50) of 2 to 4 nM against laboratory-adapted HIV-1 strain SF33 and an EC50 of 7 to 14 nM against nucleoside reverse transcriptase inhibitor-resistant HIV-1 strain 7391 with a therapeutic index of >1 × 10(4). A panel of five representative clinical virus isolates of different subtypes circulating predominantly in China was highly sensitive to WPR-6, with EC50s ranging from 1 to 6 nM. In addition, WPR-6 showed excellent antiviral potency against the most prevalent NNRTI-resistant viruses containing the K103N and Y181C mutations. To determine whether WPR-6 selects for novel resistant mutants, in vitro resistance selection was conducted with laboratory-adapted HIV-1 strain SF33 on MT-4 cells. The results demonstrated that V106I and Y188L were the two dominant NNRTI-associated resistance mutations detected in the breakthrough viruses. Taken together, these in vitro data indicate that WPR-6 has greater efficacy than the reference HEPT analogue TNK651 and the marketed drug nevirapine against HIV-1. However, to develop it as a new NNRTI, further improvement of its pharmacological properties is warranted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.